KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy

ABSTRACT

The present invention provides polypeptides, peptide dimers, and multimeric complexes comprising at least one binding moiety for KDR or VEGF/KDR complex, which have a variety of uses wherever treating, detecting, isolating or localizing angiogenesis is advantageous. Particularly disclosed are synthetic, isolated polypeptides capable of binding KDR or VEGF/KDR complex with high affinity (e.g., having a K D &lt;1 μM), and dimer and multimeric constructs comprising these polypeptides, particularly contrast agents. Also provided are methods for monitoring and evaluating the therapeutic effectiveness of treatment protocols for diseases associated with angiogenesis or endothelial cell hyperproliferation, such as cancer, using contrast agents of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/480,578 filed on Jun. 8, 2009 which is a continuation of U.S.application Ser. No. 10/661,156 filed on Sep. 11, 2003, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 10/382,082filed on Mar. 3, 2003, now abandoned, and a continuation-in-part ofInternational Application No. PCT/US03/06731 filed on Mar. 3, 2003. U.S.application Ser. No. 10/382,082 and International Application No.PCT/US03/06731 both claim the benefit of U.S. Provisional ApplicationNo. 60/360,851 filed on Mar. 1, 2002 and U.S. Provisional ApplicationNo. 60/440,411 filed on Jan. 15, 2003. The contents of the aboveapplications are incorporated herein by reference.

This application is also a continuation-in-part of U.S. application Ser.No. 11/954,130 filed on Dec. 11, 2007, which is a continuation-in-partof U.S. Application Ser. No. 11,608,395, filed Dec. 8, 2006, now U.S.Pat. No. 7,794,693, which claims priority to and benefit of U.S.Provisional Application No. 60/833,342, filed Jul. 25, 2006 and U.S.Provisional Application No. 60/749,240, filed Dec. 9, 2005, and is acontinuation-in-part of U.S. application Ser. No. 10/661,156, filed Sep.11, 2003, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 10/382,082, filed Mar. 3, 2003, now abandoned and acontinuation in-part of International Application No. PCT/US03/06731,filed Mar. 3, 2003, both of which claim priority to and benefit of U.S.Provisional Application No. 60/440,411, filed Jan. 15, 2003; and U.S.Provisional Application No. 60/360,851, filed Mar. 1, 2002. The contentsof the above applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to polypeptides and compositions usefulfor detecting and targeting primary receptors on endothelial cells forvascular endothelial growth factor (VEGF), i.e., vascular endothelialgrowth factor receptor-2 (VEGFR-2, also known as kinase domain region(KDR) and fetal liver kinase-1 (Flk-1)), and for imaging and targetingcomplexes formed by VEGF and KDR. The involvement of VEGF and KDR inangiogenesis makes the VEGF/KDR and KDR binding polypeptides of thepresent invention particularly useful for imaging important sites ofangiogenesis, e.g., neoplastic tumors, for targeting substances, e.g.,therapeutics, including radiotherapeutics, to such sites, and fortreating certain disease states, including those associated withinappropriate angiogenesis. The present invention also relates totargeting vector-phospholipid conjugates and particularly targetingpeptide-phospholipid conjugates, which target KDR and are useful intherapeutic and diagnostic compositions and methods of preparation ofthe same. The invention includes targeted ultrasound contrast agents,and particularly targeted microbubbles which include such targetingvector-phospholipid conjugates.

BACKGROUND OF THE INVENTION

In the developing embryo, the primary vascular network is established byin situ differentiation of meso-dermal cells in a process calledvasculogenesis. After embryonic vasculogenesis however, it is believedthat all subsequent generation of new blood vessels, in the embryo or inadults, is governed by the sprouting or splitting of new capillariesfrom the pre-existing vasculature in a process called angiogenesis(Pepper, M. et al., 1996. Enzyme Protein, 49:138-162; Risau, W., 1997.Nature, 386:671-674). Angiogenesis is not only involved in embryonicdevelopment and normal tissue growth and repair, it is also involved inthe female reproductive cycle, establishment and maintenance ofpregnancy, and in repair of wounds and fractures. In addition toangiogenesis that takes place in the normal individual, angiogenicevents are involved in a number of pathological processes, notably tumorgrowth and metastasis, and other conditions in which blood vesselproliferation is increased, such as diabetic retinopathy, psoriasis andarthropathies. Angiogenesis is so important in the transition of a tumorfrom hyperplastic to neoplastic growth, that inhibition of angiogenesishas shown promise as a cancer therapy (Kim, K. et al., 1993. Nature,362:841-844).

Tumor-induced angiogenesis is thought to depend on the production ofpro-angiogenic growth factors by the tumor cells, which overcome otherforces that tend to keep existing vessels quiescent and stable. The bestcharacterized of these pro-angiogenic agents or growth factors isvascular endothelial growth factor (VEGF) (Cohen et al., FASEB J., 13:9-22 (1999)). VEGF is produced naturally by a variety of cell types inresponse to hypoxia and some other stimuli. Many tumors also producelarge amounts of VEGF, and/or induce nearby stromal cells to make VEGF(Fukumura et al., Cell, 94: 715-725 (1998)). VEGF, also referred to asVEGF-A, is synthesized as five different splice isoforms of 121, 145,165, 189, and 206 amino acids. VEGF121 and VEGF165 are the main formsproduced, particularly in tumors (see Cohen et al. 1999, supra). VEGF121lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and doesnot bind to heparin or extracellular matrix, unlike VEGF165. Each of thereferences cited in this paragraph is incorporated by reference in itsentirety.

VEGF family members act primarily by binding to receptor tyrosinekinases. In general, receptor tyrosine kinases are glycoproteins havingan extracellular domain capable of binding one or more specific growthfactors, a transmembrane domain (usually an alpha helix), ajuxtamembrane domain (where the receptor may be regulated, e.g., byphosphorylation), a tyrosine kinase domain (the catalytic component ofthe receptor), and a carboxy-terminal tail, which in many receptors isinvolved in recognition and binding of the substrates for the tyrosinekinase. There are three endothelial cell-specific receptor tyrosinekinases known to bind VEGF:VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), andVEGFR-3 (Flt4). Flt-1 and KDR (also known as VEGFR-2 or Flk-1, which areused interchangeably herein) have been identified as the primary highaffinity VEGF receptors. While Flt-1 has higher affinity for VEGF, KDRdisplays more abundant endothelial cell expression (Bikfalvi et al., J.Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominatethe angiogenic response and is therefore of greater therapeutic anddiagnostic interest (see Cohen et al. 1999, supra). Expression of KDR ishighly upregulated in angiogenic vessels, especially in tumors thatinduce a strong angiogenic response (Veikkola et al., Cancer Res., 60:203-212 (2000)). The critical role of KDR in angiogenesis is highlightedby the complete lack of vascular development in homozygous KDR knockoutmouse embryos (Folkman et al., Cancer Medicine, 5th Edition (B.C. DeckerInc.; Ontario, Canada, 2000) pp. 132-152).

KDR (kinase domain region) is made up of 1336 amino acids in its matureform. The glycosylated form of KDR migrates on an SDS-PAGE gel with anapparent molecular weight of about 205 kDa. KDR contains sevenimmunoglobulin-like domains in its extracellular domain, of which thefirst three are the most important in VEGF binding (Cohen et al. 1999,supra). VEGF itself is a homodimer capable of binding to two KDRmolecules simultaneously. The result is that two KDR molecules becomedimerized upon binding and autophosphorylate, becoming much more active.The increased kinase activity in turn initiates a signaling pathway thatmediates the KDR-specific biological effects of VEGF.

Thus, not only is the VEGF binding activity of KDR in vivo critical toangiogenesis, but the ability to detect KDR upregulation on endothelialcells or to detect VEGF/KDR binding complexes would be extremelybeneficial in detecting or monitoring angiogenesis with particulardiagnostic applications such as detecting malignant tumor growth. Itwould also be beneficial in therapeutic applications such as targetingtumorcidal agents or angiogenesis inhibitors to a tumor site ortargeting agonists of KDR, VEGF/KDR, or angiogenesis to a desired site.

It is well known that gas filled ultrasound contrast agents areexceptionally efficient ultrasound reflectors for echography. Suchultrasound contrast agents include, for example, gas-filledmicrovesicles such as gas-filled microbubbles and gas filledmicroballoons. Gas filled microbubbles are particularly preferredultrasound contrast agents. (In this disclosure the term of“microbubble” specifically designates a gaseous bubble surrounded orstabilized by phospholipids). For instance injecting into thebloodstream of living bodies suspensions of air- or gas-filledmicrobubbles in a carrier liquid will strongly reinforce ultrasonicechography imaging, thus aiding in the visualization of internalanatomical structures. Imaging of vessels and internal organs canstrongly help in medical diagnosis, for instance for the detection ofneoplastic, cardiovascular and other diseases.

For both diagnostic and therapeutic purposes it would be particularlybeneficial to incorporate into gas filled ultrasound contrast agents,targeting vector compositions which exhibit high binding affinity for adesired target (such as, for example, KDR or the VEGF/KDR complex). Forexample, targeting vector-phospholipid conjugates and particularlytargeting peptide-phospholipid conjugates may be used to preparetargeted, gas filled ultrasound contrast agents. In addition, it wouldbe particularly beneficial to have methods for large scale production ofhighly purified forms of such targeting vector-phospholipid conjugates.Such compositions and methods would allow for production of compositionsfor use in diagnostic or therapeutic applications such as, for example,precise targeting of reporter moieties, tumoricidal agents orangiogenesis inhibitors to the target site.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides and compositions usefulfor detecting and targeting primary receptors on endothelial cells forvascular endothelial growth factor (VEGF), i.e., vascular endothelialgrowth factor receptor-2 (VEGFR-2, also known as kinase domain region(KDR) and fetal liver kinase-1 (Flk-1)), and for imaging and targetingcomplexes formed by VEGF and KDR. The involvement of VEGF and KDR inangiogenesis makes the VEGF/KDR and KDR binding polypeptides of thepresent invention particularly useful for imaging important sites ofangiogenesis, e.g., neoplastic tumors, for targeting substances, e.g.,therapeutics, including radiotherapeutics, to such sites, and fortreating certain disease states, including those associated withinappropriate angiogenesis.

A group of polypeptides has been discovered that bind to KDR or VEGF/KDRcomplex (referred to herein as “KDR binding polypeptides” or “KDRbinding moieties” and homologues thereof). Such KDR and VEGF/KDR bindingpolypeptides will concentrate at the sites of angiogenesis, thusproviding a means for detecting and imaging sites of activeangiogenesis, which can include sites of neoplastic tumor growth. SuchKDR and VEGF/KDR binding polypeptides provide novel therapeutics toinhibit or promote, e.g., angiogenesis. The preparation, use andscreening of such polypeptides, for example as imaging agents or asfusion partners for KDR or VEGF/KDR-homing therapeutics, is described indetail herein.

In answer to the need for improved materials and methods for detecting,localizing, measuring and possibly affecting (inhibiting or enhancing),e.g., angiogenesis, it has been surprisingly discovered that sevenfamilies of non-naturally occurring polypeptides bind specifically toKDR or VEGF/KDR complex. Appropriate labeling of such polypeptidesprovides detectable imaging agents that can bind, e.g., at highconcentration, to KDR-expressing endothelial cells or cells exhibitingVEGF/KDR complexes, providing angiogenesis-specific imaging agents. TheKDR and VEGF/KDR binding polypeptides of the instant invention can thusbe used in the detection and diagnosis of such angiogenesis-relateddisorders. Conjugation or fusion of such polypeptides with effectiveagents such as VEGF inhibitors or tumorcidal agents can also be used totreat pathogenic tumors, e.g., by causing the conjugate or fusion to“home” to the site of active angiogenesis, thereby providing aneffective means for treating pathogenic conditions associated withangiogenesis.

This invention pertains to KDR and VEGF/KDR binding polypeptides, andincludes use of a single binding polypeptide as a monomer or in amultimeric or polymeric construct as well as use of more than onebinding polypeptide of the invention in multimeric or polymericconstructs. Binding polypeptides according to this invention are usefulin any application where binding, detecting or isolating KDR or VEGF/KDRcomplex, or fragments thereof retaining the polypeptide binding site, isadvantageous. A particularly advantageous use of the bindingpolypeptides disclosed herein is in a method of imaging angiogenesis invivo. The method entails the use of specific binding polypeptidesaccording to the invention for detecting a site of angiogenesis, wherethe binding polypeptides have been detectably labeled for use as imagingagents, including magnetic resonance imaging (MRI) contrast agents,x-ray imaging agents, radiopharmaceutical imaging agents, ultrasoundimaging agents, and optical imaging agents.

Another advantageous use of the KDR and VEGF/KDR complex bindingpolypeptides disclosed herein is to target therapeutic agents (includingcompounds capable of providing a therapeutic, radiotherapeutic orcytotoxic effect), or delivery vehicles for therapeutics (includingdrugs, genetic material, etc.) to sites of angiogenesis or other tissueexpressing KDR.

Constructs comprising two or more KDR or KDR/VEGF binding polypeptidesshow improved ability to bind the target molecule compared to thecorresponding monomeric binding polypeptides. For example, as shown inExperiment D of Example 5, tetrameric constructs of KDR bindingpolypeptides provided herein showed improved ability to bindKDR-transfected 293H cells. Combining two or more binding polypeptidesin a single molecular construct appears to improve the avidity of theconstruct over the monomeric binding polypeptides as shown by a decreasein K_(D).

In addition, as demonstrated herein, constructs comprising two or morebinding polypeptides specific for different epitopes of KDR and/orKDR/VEGF (e.g., “heteromeric” or “heteromultimeric” constructs, see U.S.Application No. 60/440,201, U.S. application Ser. No. 10/379,287, filedMar. 3, 2003, and U.S. application Ser. No. 10/661,032 by ChristopheArbogast et al., filed Sep. 11, 2003, the contents of which areincorporated herein) were made. Constructs comprising two or morebinding polypeptides provided herein are expected to bind to multiplesites on KDR or VEGF/KDR. The heteromeric constructs show superiorbinding ability over both the corresponding monomers and multimericconstructs comprising multiple copies of the same binding polypeptide.Furthermore, heteromeric constructs comprising two or more bindingpeptides specific for different epitopes, together with a controlpeptide, were also able to efficiently bind KDR-transfected 293H cells.Thus, inclusion of two or more binding polypeptides that recognizedifferent epitopes further improves the avidity of the construct for thetarget molecule, as demonstrated by a decrease in K_(D).

Heteromeric constructs of the binding polypeptides provided herein showimproved ability to inhibit receptor tyrosine kinase function. Based onexperiments described herein, dimeric and other multimeric constructs ofthe present invention comprising at least two binding polypeptidesspecific for different epitopes of KDR and/or KDR/VEGF complex areexpected to inhibit the function of receptor tyrosine kinases. Inparticular, such constructs are expected to inhibit the function ofVEGFR-2/KDR, VEGFR-1/Flt-1 and VEGFR-3/Flt-4.

For the purposes of the present invention, receptor tyrosine kinasefunction can include any one of: oligomerization of the receptor,receptor phosphorylation, kinase activity of the receptor, recruitmentof downstream signaling molecules, induction of genes, induction of cellproliferation, induction of cell migration, or combination thereof. Forexample, heteromeric constructs of binding polypeptides provided hereininhibit VEGF-induced KDR receptor activation in human endothelial cells,demonstrated by the inhibition of VEGF-induced phosphorylation of theKDR receptor. In addition, heteromeric constructs of binding peptidesprovided herein inhibit VEGF-stimulated endothelial cell migration. Asshown herein, targeting two or more distinct epitopes on KDR with asingle binding construct greatly improves the ability of the constructto inhibit receptor function. Even binding peptides with weak ability toblock receptor activity can be used to generate heteromeric constructshaving improved ability to block VEGF-induced receptor function.

Therefore, the present invention also is drawn to constructs comprisingtwo or more binding polypeptides. In one embodiment, the multimericconstructs comprise two or more copies of a single binding polypeptide.In another embodiment, the multimeric constructs of the presentinvention comprise two or more binding polypeptides, such that at leasttwo of the binding polypeptides in the construct are specific fordifferent epitopes of KDR and/or KDR/VEGF. These constructs are alsoreferred to herein as “heteromeric constructs,” “heteromultimers,” etc.The constructs of the present invention can also include unrelated, orcontrol peptide(s). The constructs can include two or more, three ormore, or four or more binding polypeptides. Based on the teachingsprovided herein, one of ordinary skill in the art is able to assemblethe binding polypeptides provided herein into multimeric constructs andto select multimeric constructs having improved properties, such asimproved ability to bind the target molecule, or improved ability toinhibit receptor tyrosine kinase function. Such multimeric constructshaving improved properties are included in the present invention.

Consensus sequences 1-14 have been determined based on the specific KDRand VEGF/KDR binding polypeptides shown in Tables 1-7. In specificembodiments, KDR and VEGF/KDR binding polypeptides of the inventioncomprise one or more of these sequences. Such preferred KDR or VEGF/KDRcomplex binding polypeptides include polypeptides with the potential toform a cyclic or loop structure between invariant cysteine residuescomprising, or alternatively consisting of, an amino acid sequenceselected from the group consisting of Consensus Sequences 1-5 below:

Consensus Sequence 1: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄(TN8), wherein

X₁ is Ala, Arg, Asp, Gly, His, Leu, Lys, Pro, Ser, Thr, Trp, Tyr or Val;

X₂ is Asn, Asp, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr or Val;

X₃ is Asn, Asp, Gln, Glu, Ile, Leu, Met, Thr, Trp or Val;

X₅ is Ala, Arg, Asn, Asp, Gln, Glu, His, Ile, Lys, Phe, Pro, Ser, Trp orTyr;

X₆ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr or Val;

X₇ is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr or Val;

X₈ is Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Thr, Trp or Tyr;

X₉ is Arg, Gln, Glu, Gly, Ile, Leu, Met, Pro, Thr, Trp, Tyr or Val;

X₁₀ is Ala, Arg, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Trp orTyr;

X₁₂ is Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val;

X₁₃ is Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,Thr, Trp or Tyr; and

X₁₄ is Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trpor Tyr,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 2:X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (TN12),wherein

X₁ is Ala, Asn, Asp, Gly, Leu, Pro, Ser, Trp or Tyr (preferably Asn,Asp, Pro or Tyr);

X₂ is Ala, Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr(preferably Asp, Gly, Pro, Ser or Trp);

X₃ is Ala, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Ser, Thr,Trp, Tyr or Val (preferably Trp);

X₅ is Arg, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Thr, Trp, Tyr or Val(preferably Glu, Ile or Tyr);

X₆ is Ala, Arg, Asn, Cys, Glu, Ile, Leu, Met, Phe, Ser, Trp or Tyr(preferably Glu, Phe or Tyr);

X₇ is Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Pro, Ser, Thr, Trp, Tyr orVal (preferably Glu);

X₈ is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Phe, Pro, Ser, Trp, Tyr orVal (preferably Gln or Ser);

X₉ is Asp, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp orTyr (preferably Asp);

X₁₀ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val (preferably Lys or Ser);

X₁₁ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Trp, Tyr or Val(preferably Gly or Tyr);

X₁₂ is Ala, Arg, Gln, Gly, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, Tyror Val (preferably Trp or Thr);

X₁₃ is Arg, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, Trp or Val(preferably Glu or Trp);

X₁₄ is Arg, Asn, Asp, Glu, His, Ile, Leu, Met, Phe, Pro, Thr, Trp, Tyror Val (preferably Phe);

X₁₆ is Ala, Asn, Asp, Gln, Glu, Gly, Lys, Met, Phe, Ser, Thr, Trp, Tyror Val (preferably Asp);

X₁₇ is Arg, Asn, Asp, Cys, Gly, His, Phe, Pro, Ser, Trp or Tyr(preferably Pro or Tyr); and

X₁₈ is Ala, Asn, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp or Tyr(preferably Asn, Pro or Trp),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 3: X₁-X₂-X₃-Cys-X₅-X₆-X₇-Gly-X₉-Cys-X₁₁-X₁₂-X₁₃(TN7), wherein

X₁ is Gly or Trp;

X₂ is Ile, Tyr or Val;

X₃ is Gln, Glu Thr or Trp;

X₅ is Asn, Asp or Glu;

X₆ is Glu, His, Lys or Phe;

X₇ is Asp, Gln, Leu, Lys Met or Tyr;

X₉ is Arg, Gln, Leu, Lys or Val;

X₁₁ is Arg, Phe, Ser, Trp or Val;

X₁₂ is Glu, His or Ser; and

X₁₃ is Glu, Gly, Trp or Tyr,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 4:X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅ (TN9), wherein

X₁ is Arg, Asp, Gly, Ile, Met, Pro or Tyr (preferably Tyr);

X₂ is Asp, Gly, His, Pro or Trp (preferably Gly or Trp);

X₃ is Gly, Pro, Phe, Thr or Trp (preferably Pro);

X₅ is Ala, Asp, Lys, Ser, Trp or Val (preferably Lys);

X₆ is Asn, Glu, Gly, His or Leu;

X₇ is Gln, Glu, Gly, Met, Lys, Phe, Tyr or Val (preferably Met);

X₈ is Ala, Asn, Asp, Gly, Leu, Met, Pro, Ser or Thr;

X₉ is His, Pro or Trp (preferably Pro);

X₁₀ is Ala, Gly, His, Leu, Trp or Tyr (preferably His or Trp);

X₁₁ is Ala, Asp, Gln, Leu, Met, Thr or Trp;

X₁₃ is Ala, Lys, Ser, Trp or Tyr (preferably Trp);

X₁₄ is Asp, Gly, Leu, His, Met, Thr, Trp or Tyr (preferably His, Trp, orTyr); and

X₁₅ is Asn, Gln, Glu, Leu, Met, Pro or Trp (preferably Glu, Met or Trp),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 5:X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-Ser-Gly-Pro-X₁₂-X₁₃-X₁₄-X₁₅-Cys-X₁₇-X₁₈-X₁₉(MTN13; SEQ ID NO: 1), wherein

X₁ is Arg, Glu, His, Ser or Trp;

X₂ is Asn, Asp, Leu, Phe, Thr or Val;

X₃ is Arg, Asp, Glu, His, Lys or Thr;

X₅ is Asp, Glu, His or Thr;

X₆ is Arg, His, Lys or Phe;

X₇ is Gln, Ile, Lys, Tyr or Val;

X₈ is Gln, Ile, Leu, Met or Phe;

X₁₂ is Asn, Asp, Gly, His or Tyr;

X₁₃ is Gln, Gly, Ser or Thr;

X₁₄ is Glu, Lys, Phe or Ser;

X₁₅ is Glu, Ile, Ser or Val;

X₁₇ is Glu, Gly, Lys, Phe, Ser or Val;

X₁₈ is Arg, Asn, Ser or Tyr; and

X₁₉ is Asp, Gln, Glu, Gly, Met or Tyr,

and wherein the polypeptide binds KDR or a VEGF/KDR complex.

Further analysis of the polypeptides isolated from the TN8 library (seeConsensus Sequence 1) revealed sub-families of preferred bindingpolypeptides, which are described by the Consensus Sequences 6, 7 and 8as follows:

Consensus Sequence 6: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-Tyr-Cys-X₁₂-X₁₃-X₁₄,wherein

X₁ is Ala, Arg, Asp, Leu, Lys, Pro, Ser or Val;

X₂ is Asn, Asp, Glu, Lys, Thr or Ser (preferably Asn, Asp, Glu or Lys);

X₃ is Ile, Leu or Trp;

X₅ is Ala, Arg, Glu, Lys or Ser (preferably Glu);

X₆ is Ala, Asp, Gln, Glu, Thr or Val (preferably Asp or Glu);

X₇ is Asp or Glu;

X₈ is Tip or Tyr;

X₉ is Thr or Tyr (preferably Tyr);

X₁₂ is Glu, Met, Phe, Trp or Tyr (preferably Trp, Phe, Met, or Tyr);

X₁₃ is Ile, Leu or Met; and

X₁₄ is Ile, Leu, Met, Phe or Thr (preferably Thr or Leu),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 7:Trp-Tyr-Trp-Cys-X₅-X₆-X₇-Gly-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄ (SEQ ID NO: 2),wherein

X₅ is Asp, Gln or His;

X₆ is His or Tyr (preferably Tyr);

X₇ is Ile, His or Tyr;

X₉ is Ile, Met or Val;

X₁₀ is Gly or Tyr;

X₁₂ is Asp, Lys or Pro;

X₁₃ is Gln, Gly or Trp; and

X₁₄ is Phe, Ser or Thr,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 8: X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-Gly-X₁₀-Cys-X₁₂-X₁₃-X₁₄,wherein

X₁ is Gly, Leu, His, Thr, Trp or Tyr (preferably Trp, Tyr, Leu or His);

X₂ is Ile, Leu, Thr, Trp or Val (preferably Val, Ile or Leu);

X₃ is Asp, Glu, Gln, Trp or Thr, (preferably Glu, Asp or Gln);

X₅ is Ala, Arg, Asn, Asp, His, Phe, Trp or Tyr (preferably Tyr, Trp orPhe);

X₆ is Ala, Asp, Gln, His, Lys, Met, Ser, Thr, Trp, Tyr or Val;

X₇ is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr orVal;

X₈ is Asp, Phe, Ser, Thr, Trp or Tyr (preferably Thr, Ser or Asp);

X₁₀ is Ala, Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr(preferably Arg or Lys);

X₁₂ is Arg, Gln, His, Ile, Lys, Met, Phe, Thr, Trp, Tyr or Val(preferably Tyr, Trp, Phe, Ile or Val);

X₁₃ is Arg, Asn, Asp, Glu, His, Met, Pro, Ser or Thr; and

X₁₄ is Arg, Gln, Glu, Gly, Phe, Ser, Trp or Tyr,

and wherein the polypeptide binds KDR or a VEGF/KDR complex.

Further analysis of the polypeptides isolated from the TN12 library (seeConsensus Sequence 2) revealed sub-families of preferred bindingpolypeptides, which are described by Consensus Sequences 9-12 and 9A asfollows:

Consensus Sequence 9:X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-Trp-Gly-Gly-X₁₂-X₁₃-Cys-X₁₅-X₁₆-X₁₇ (SEQ ID NO:3)(TN11, i.e., 11-mer binders isolated from the TN12 library), wherein

X₁ is Ser, Phe, Trp, Tyr or Gly (preferably Ser);

X₂ is Arg, Gly, Ser or Trp (preferably Arg);

X₃ is Ala, Glu, Ile or Val (preferably Val or Ile);

X₅ is Ala, Phe or Trp (preferably Trp or Phe);

X₆ is Glu or Lys (preferably Glu);

X₇ is Asp, Ser, Trp or Tyr (preferably Asp, Trp or Tyr);

X₈ is Phe, Pro or Ser (preferably Ser);

X₁₂ is Gln or Glu (preferably Glu);

X₁₃ is Ile, Phe or Val;

X₁₅ is Gln, Ile, Leu, Phe or Tyr (preferably Phe, Tyr or Leu);

X₁₆ is Arg, Gly or Pro (preferably Arg); and

X₁₇ is Gln, His, Phe, Ser, Tyr or Val (preferably Tyr, Phe, His or Val),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 9A:X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-Cys-X₁₅-X₁₆-X₁₇ (TN11, i.e.,11-mer binders isolated from the TN12 library; SEQ ID NO: 3), wherein

X₁ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val;

X₂ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Ser,Thr, Trp, Tyr or Val;

X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Thr, Trp, Tyr or Val;

X₅ is Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,Trp, Tyr or Val;

X₆ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp or Tyr;

X₇ is Ala, Arg, Asp, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser,Thr, Trp, Tyr or Val;

X₈ is Ala, Arg, Asp, Asn, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr or Val;

X₉ is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Phe, Pro, Ser, Trp or Tyr;

X₁₀ is Asp, Gln, Glu, Gly, His, Ile, Leu, Phe, Ser, Thr, Trp, Tyr orVal;

X₁₁ is Ala, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp,Tyr or Val;

X₁₂ is Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr or Val;

X₁₃ is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val;

X₁₅ is Ala, Asp, Asn, Glu, Gly, Ile, His, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val;

X₁₆ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val;

X₁₇ is Ala, Arg, Asp, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Tyr or Val,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 10:Tyr-Pro-X₃-Cys-X₅-Glu-X₇-Ser-X₉-Ser-X₁₁-X₁₂-X₁₃-Phe-Cys-X₁₆-X₁₇-X₁₈(TN12; SEQ ID NO: 4), wherein

X₃ is Gly or Trp (preferably Trp);

X₅ is His or Tyr (preferably His, or Tyr);

X₇ is His, Leu or Thr;

X₉ is Asp or Leu (preferably Asp);

X₁₁ is Gly or Val (preferably Val);

X₁₂ is Thr or Val (preferably Thr);

X₁₃ is Arg or Trp (preferably Arg);

X₁₆ is Ala or Val (preferably Val);

X₁₇ is Asp or Pro (preferably Pro); and

X₁₈ is Gly or Trp (preferably Trp),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 11:X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Gly-X₁₂-Trp-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (TN12;SEQ ID NO: 5), wherein

X₁ is Asp, Gly, Pro or Ser (preferably Asp);

X₂ is Arg, Asn, Asp, Gly or Ser (preferably Asp, Asn, or Ser);

X₃ is Gly, Thr, Trp or Tyr (preferably Trp or Tyr);

X₅ is Glu, Met or Thr (preferably Glu);

X₆ is Ile, Leu, Met or Phe (preferably Met, Leu, or Phe);

X₇ is Arg, Asp, Glu, Met, Trp or Val;

X₈ is Asn, Gln, Gly, Ser or Val;

X₉ is Asp or Glu;

X₁₀ is Lys, Ser, Thr or Val (preferably Lys);

X₁₂ is Arg, Gln, Lys or Trp (preferably Tip, Arg, or Lys);

X₁₄ is Asn, Leu, Phe or Tyr (preferably Tyr, Phe, or Asn);

X₁₆ is Gly, Phe, Ser or Tyr (preferably Tyr or Phe);

X₁₇ is Gly, Leu, Pro or Ser (preferably Pro or Ser); and

X₁₈ is Ala, Asp, Pro, Ser, Trp or Tyr,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 12:Asn-Trp-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈ (TN12;SEQ ID NO: 6), wherein

X₃ is Glu or Lys;

X₅ is Glu or Gly;

X₆ is Trp or Tyr;

X₇ is Ser or Thr;

X₈ is Asn or Gln;

X₉ is Gly or Met;

X₁₀ is Phe or Tyr;

X₁₁ is Asp or Gln;

X₁₂ is Lys or Tyr;

X₁₃ is Glu or Thr;

X₁₄ is Glu or Phe;

X₁₆ is Ala or Val;

X₁₇ is Arg or Tyr; and

X₁₈ is Leu or Pro,

and wherein the polypeptide binds KDR or a VEGF/KDR complex.

Analysis of the binding polypeptides isolated from a linear displaylibrary (Lin20) defined two families of preferred embodiments includingthe amino acid sequences of Consensus Sequences 13 and 14 as follows:

Consensus Sequence 13: Z₁-X₁-X₂-X₃-X₄-X₅-Z₂ (Lin20), wherein, Z₁ is apolypeptide of at least one amino acid or is absent;

X₁ is Ala, Asp, Gln or Glu (preferably Gln or Glu);

X₂ is Ala, Asp, Gln, Glu Pro (preferably Asp, Glu or Gln);

X₃ is Ala, Leu, Lys, Phe, Pro, Trp or Tyr (preferably Trp, Tyr, Phe orLeu);

X₄ is Asp, Leu, Ser, Trp, Tyr or Val (preferably Tyr, Trp, Leu or Val);

X₅ is Ala, Arg, Asp, Glu, Gly, Leu, Trp or Tyr (preferably Trp, Tyr orLeu); and

Z₂ is a polypeptide of at least one amino acid or is absent,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Consensus Sequence 14: X₁-X₂-X₃-Tyr-Trp-Glu-X₇-X₈-X₉-Leu (Lin20; SEQ IDNO: 7), wherein, the sequence can optionally have a N-terminalpolypeptide, C-terminal polypeptide, or a polypeptide at both termini ofat least one amino acid; and wherein

X₁ is Asp, Gly or Ser (preferably Gly);

X₂ is Ile, Phe or Tyr;

X₃ is Ala, Ser or Val;

X₇ is Gln, Glu, Ile or Val;

X₈ is Ala, Ile or Val (preferably Ile or Val);

X₉ is Ala, Glu, Val or Thr;

and wherein the polypeptide binds KDR or a VEGF/KDR complex.

Preferred embodiments comprising the Consensus Sequence 1 above, includepolypeptides in which X₃ is Trp and the amino acid sequence of X₇-X₁₀ isAsp-Trp-Tyr-Tyr (SEQ ID NO: 8). More preferred structures includepolypeptides comprising Consensus Sequence 1, wherein X₃ is Trp and theamino acid sequence of X₅-X₁₀ is Glu-Glu-Asp-Trp-Tyr-Tyr (SEQ ID NO: 9).Additional preferred polypeptides comprising Consensus Sequence 1include polypeptides in which: X₃ is Trp and the amino acid sequence ofX₅-X₁₀ is Glu-Glu-Asp-Trp-Tyr-Tyr (SEQ ID NO: 9), and the peptideX₁₃-X₁₄ is Ile-Thr. Of these preferred polypeptides, it is additionallypreferred that X₁ will be Pro and X₁₂ will be one of Phe, Trp or Tyr.

Particular embodiments of the cyclic polypeptide families describedabove are disclosed in Tables 1, 2, 4, 5, 6 and 7, infra.

Additional cyclic polypeptides found to bind a KDR or VEGF/KDR targethave a cyclic portion (or loop), formed by a disulfide bond between thetwo cysteine residues, consisting of ten amino acids, for example, asfollows:

(SEQ ID NO: 10) Asn-Asn-Ser-Cys-Trp-Leu-Ser-Thr-Thr-Leu-Gly-Ser-Cys-Phe-Phe-Asp, (SEQ ID NO: 11)Asp-His-His-Cys-Tyr-Leu-His-Asn-Gly-Gln-Trp-Ile- Cys-Tyr-Pro-Phe,(SEQ ID NO: 12) Asn-Ser-His-Cys-Tyr-Ile-Trp-Asp-Gly-Met-Trp-Leu-Cys-Phe-Pro-Asp.

Additional preferred embodiments include linear polypeptides capable ofbinding a KDR or VEGF/KDR target comprising, or alternatively consistingof, a polypeptide having an amino acid sequence selected from the groupof amino acid sequences set forth in Table 3, infra.

The polypeptides of the invention can optionally have additional aminoacids attached at either or both of the N- and C-terminal ends. Inpreferred embodiments, binding polypeptides according to the inventioncan be prepared having N-terminal and/or C-terminal flanking peptides ofone or more, preferably two, amino acids corresponding to the flankingpeptides of the display construct of the phage selectant from which thebinding polypeptides were isolated. Preferred amino-terminal flankingpeptides include Ala-Gly- (most preferably for TN7, TN8 and TN9sequences), Gly-Ser- (most preferably for TN10 sequences), Gly-Asp-(mostpreferably for TN12 sequences), Ala-Gln- (most preferably for linearsequences), and Ser-Gly- (most preferably for MTN13 sequences).Preferred carboxy-terminal flanking peptides include -Gly-Thr (mostpreferably for TN7, TN8, TN9 sequences), -Ala-Pro (most preferably forTN10 sequences), -Asp-Pro (most preferably for TN12 sequences), -Gly-Gly(most preferably for linear sequences), and -Gly-Ser (most preferablyfor MTN13 sequences). Single terminal amino acids can also be added tothe binding polypeptides of the invention, and preferred terminal aminoacids will preferably correspond to the parental phage displayconstruct, e.g., most preferably, N-terminal amino acids will beselected from Gly- (most preferably for TN7, TN8, TN9, MTN13 sequences),Ser- (most preferably for TN10 sequences), Asp- (most preferably forTN12 sequences), and Gln- (most preferably for linear sequences), andmost preferably C-terminal amino acids will be selected from -Gly (mostpreferably for TN7, TN8, TN9, MTN13 and linear sequences), -Ala (mostpreferably for TN10 sequences), and -Asp (most preferably for TN12sequences). Conservative substitutions (i.e., substitute amino acidsselected within the following groups: {Arg, His, Lys}, {Glu, Asp}, {Asn,Cys, Glu, Gly, Ser, Thr, Tyr}, {Ala, Ile, Leu, Met, Phe, Pro, Trp, Val})for such flanking amino acids are also contemplated.

Examination of the sequence information and binding data from theisolates of libraries containing polypeptides with the potential to formloop structures (e.g., libraries designated TN7, TN8, TN9, TN10, TN12and MTN13) identifies a series of KDR or VEGF/KDR complex bindingpolypeptides that may form loop structures. In specific embodiments,cyclic KDR— or VEGF/KDR— binding polypeptides of the invention comprise,or alternatively, consist of, an amino acid sequence selected from LoopConsensus Sequences 15-19 as follows:

Loop Consensus Sequence 15: Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys (TN8), wherein

X₂ is Ala, Arg, Asn, Asp, Gln, Glu, His, Ile, Lys, Phe, Pro, Ser, Trp orTyr (preferably Asp, Glu or Tyr);

X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr or Val (preferably Glu, Met or Tyr);

X₄ is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr or Val (preferably Asp);

X₅ is Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Thr, Trp or Tyr(preferably Trp or Thr);

X₆ is Arg, Gln, Glu, Gly, Ile, Leu, Met, Pro, Thr, Trp, Tyr or Val(preferably Gly or Tyr); and

X₇ is Ala, Arg, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr(preferably Lys or Tyr),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Loop Consensus Sequence 16: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys(TN12), wherein

X₂ is Arg, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Thr, Trp, Tyr or Val(preferably Glu, Ile or Tyr);

X₃ is Ala, Arg, Asn, Cys, Glu, Ile, Leu, Met, Phe, Ser, Trp or Tyr(preferably Glu, Phe or Tyr);

X₄ is Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Pro, Ser, Thr, Trp, Tyr orVal (preferably Glu);

X₅ is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Phe, Pro, Ser, Trp, Tyr orVal (preferably Gln or Ser);

X₆ is Asp, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp orTyr (preferably Asp);

X₇ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val (preferably Lys or Ser);

X₈ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Trp, Tyr or Val(preferably Gly or Tyr);

X₉ is Ala, Arg, Gln, Gly, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, Tyr orVal (preferably Trp or Thr);

X₁₀ is Arg, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, Trp or Val(preferably Glu or Trp); and

X₁₁ is Arg, Asn, Asp, Glu, His, Ile, Leu, Met, Phe, Pro, Thr, Trp, Tyror Val (preferably Phe),

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Loop Consensus Sequence 17: Cys-X₂-X₃-X₄-Gly-X₆-Cys (TN7), wherein

X₂ is Asn, Asp or Glu;

X₃ is Glu, His, Lys or Phe;

X₄ is Asp, Gln, Leu, Lys, Met or Tyr; and

X₆ is Arg, Gln, Leu, Lys or Val,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Loop Consensus Sequence 18: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys (TN9), wherein

X₂ is Ala, Asp, Lys, Ser, Trp or Val (preferably Lys);

X₃ is Asn, Glu, Gly, His or Leu;

X₄ is Gln, Glu, Gly, Met, Lys, Phe, Tyr or Val (preferably Met);

X₅ is Ala, Asn, Asp, Gly, Leu, Met, Pro, Ser or Thr;

X₆ is His, Pro or Trp (preferably Pro or Trp);

X₇ is Ala, Gly, His, Leu, Trp or Tyr (preferably Trp); and

X₈ is Ala, Asp, Gln, Leu, Met, Thr or Trp,

and wherein the polypeptide binds KDR or a VEGF/KDR complex; or

Loop Consensus Sequence 19:Cys-X₂-X₃-X₄-X₅-Ser-Gly-Pro-X₉-X₁₀-X₁₁-X₁₂-Cys (MTN13; SEQ ID NO: 13),wherein

X₂ is Asp, Glu, His or Thr;

X₃ is Arg, His, Lys or Phe;

X₄ is Gln, Ile, Lys, Tyr or Val;

X₅ is Gln, Ile, Leu, Met or Phe;

X₉ is Asn, Asp, Gly, His or Tyr;

X₁₀ is Gln, Gly, Ser or Thr;

X₁₁ is Glu, Lys, Phe or Ser; and

X₁₂ is Glu, Ile, Ser or Val,

and wherein the polypeptide binds KDR or a VEGF/KDR complex.

Preferred embodiments of the cyclic peptides of Loop Consensus Sequence15 include KDR and/or VEGF/KDR complex binding polypeptides comprisingLoop Consensus Sequences 20-22 as follows:

Loop Consensus Sequence 20: Cys-X₂-X₃-X₄-X₅-X₆-Tyr-Cys (TN8), wherein

X₂ is Ala, Arg, Glu, Lys or Ser (preferably Glu);

X₃ is Ala, Asp, Gln, Glu, Thr or Val (preferably Asp or Glu);

X₄ is Asp or Glu;

X₅ is Tip or Tyr; and

X₆ is Thr or Tyr (preferably Tyr); or

Loop Consensus Sequence 21: Cys-X₂-X₃-X₄-Gly-X₆-X₇-Cys (TN8), wherein

X₂ is Asp, Gln or His;

X₃ is His or Tyr (preferably Tyr);

X₄ is His, Ile or Tyr;

X₆ is Ile, Met or Val; and

X₇ is Gly or Tyr; or

Loop Consensus Sequence 22: Cys-X₂-X₃-X₄-X₅-Gly-X₇-Cys (TN8), wherein

X₂ is Ala, Arg, Asn, Asp, His, Phe, Tip or Tyr (preferably Tyr, Trp orPhe);

X₃ is Ala, Asp, Gln, His, Lys, Met, Ser, Thr, Trp, Tyr or Val;

X₄ is Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr orVal;

X₅ is Asp, Phe, Ser, Thr, Trp or Tyr (preferably Thr, Ser or Asp); and

X₇ is Ala, Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr(preferably Arg or Lys).

Preferred embodiments of the cyclic peptides of Loop Consensus Sequence16 include KDR and/or VEGF/KDR complex binding polypeptides comprisingsequences of Loop Consensus Sequences 23-26 as follows:

Loop Consensus Sequence 23: Cys-X₂-X₃-X₄-X₅-Trp-Gly-Gly-X₉-X₁₀-Cys(TN11, i.e., 11-mers based on isolates of the TN12 library; SEQ ID NO:14), wherein

X₂ is Ala, Phe or Trp (preferably Trp or Phe);

X₃ is Glu or Lys (preferably Glu);

X₄ is Asp, Ser, Trp or Tyr (preferably Asp, Trp or Tyr);

X₅ is Phe, Pro or Ser (preferably Ser);

X₉ is Gln or Glu (preferably Glu); and

X₁₀ is Ile, Phe or Val; or

Loop Consensus Sequence 24: Cys-X₂-Glu-X₄-Ser-X₆-Ser-X₈-X₉-X₁₀-Phe-Cys(TN12; SEQ ID NO: 15), wherein

X₂ is His or Tyr;

X₄ is Leu, His or Thr;

X₆ is Asp or Leu (preferably Asp);

X₈ is Gly or Val (preferably Val);

X₉ is Thr or Val (preferably Thr); and

X₁₀ is Arg or Trp (preferably Arg); or

Loop Consensus Sequence 25: Cys-X₂-X₃-X₄-X₅-X₆-X₇-Gly-X₉-Trp-X₁₁-Cys(TN12; SEQ ID NO: 16), wherein

X₂ is Glu, Met or Thr (preferably Glu);

X₃ is Ile, Leu, Met or Phe (preferably Met, Leu or Phe);

X₄ is Arg, Asp, Glu, Met, Trp or Val;

X₅ is Asn, Gln, Gly, Ser or Val;

X₆ is Glu or Asp;

X₇ is Lys, Ser, Thr or Val (preferably Lys);

X₉ is Arg, Gln, Lys or Trp (preferably Trp, Arg or Lys); and

X₁₁ is Asn, Leu, Phe or Tyr (preferably Tyr, Phe or Asn); or

Loop Consensus Sequence 26: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys(TN12), wherein

X₂ is Glu or Gly;

X₃ is Trp or Tyr;

X₄ is Ser or Thr;

X₅ is Asn or Gln;

X₆ is Gly or Met;

X₇ is Phe or Tyr;

X₈ is Asp or Gln;

X₉ is Lys or Tyr;

X₁₀ is Glu or Thr; and

X₁₁ is Glu or Phe.

Preferred embodiments of the cyclic peptides of Loop Consensus Sequence17 include KDR and/or VEGF/KDR complex binding polypeptides comprisingsequences of Loop Consensus Sequence 27 as follows:

Loop Consensus Sequence 27: Cys-X₂-X₃-X₄-Gly-X₆-Cys (TN7), wherein

X₂ is Asn, Asp or Glu;

X₃ is Glu, His, Lys or Phe;

X₄ is Asp, Gln, Leu, Lys, Met or Tyr; and

X₆ is Arg, Gln, Leu, Lys or Val.

Preferred embodiments of the cyclic peptides of Loop Consensus Sequence18 include KDR and/or VEGF/KDR complex binding polypeptides comprisingsequences of Loop Consensus Sequence 28 as follows:

Loop Consensus Sequence 28: Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys (TN9), wherein

X₂ is Ala, Lys, Ser, Trp or Val (preferably Lys);

X₃ is Asn, Glu, Gly, His or Leu;

X₄ is Glu, Gly, Lys, Met or Tyr (preferably Met);

X₅ is Ala, Asn, Asp, Leu, Met, Pro or Ser;

X₆ is His, Pro or Trp (preferably Pro);

X₇ is His, Leu, Trp or Tyr (preferably Tip or His); and

X₈ is Ala, Asp, Gln, Leu, Met, Thr or Trp.

Preferred embodiments of the cyclic peptides of Loop Consensus Sequence19 include KDR and/or VEGF/KDR complex binding polypeptides comprisingsequences of Loop Consensus Sequence 29 as follows:

Loop Consensus Sequence 29:Cys-X₂-X₃-X₄-X₅-Ser-Gly-Pro-X₉-X₁₀-X₁₁-X₁₂-Cys (MTN13; SEQ ID NO: 17),wherein

X₂ is Asp, Glu, His or Thr;

X₃ is Arg, His, Lys or Phe;

X₄ is Gln, Ile, Lys, Tyr or Val;

X₅ is Gln, Ile, Leu, Met or Phe;

X₉ is Asn, Asp, Gly, His or Tyr;

X₁₀ is Gln, Gly, Ser or Thr;

X₁₁ is Glu, Lys, Phe or Ser; and

X₁₂ is Glu, Ile, Ser or Val.

Chemical or physical modifications, as well as any sequencemodifications, described herein are encompassed for use with any of thespecific sequences disclosed herein and/or any specific sequences thatconform to any of the consensus sequences described herein.

The KDR and VEGF/KDR binding polypeptides described above can optionallyhave additional amino acids attached at either or both of the N- andC-terminal ends and can be modified, optimized or employed in multimericconstructs. Further, the invention includes homologues of the KDR andVEGF/KDR complex binding peptides as defined herein.

Another aspect of the present invention relates to modifications of theforegoing polypeptides to provide specific angiogenesis imaging agentsby detectably labeling a polypeptide according to the present invention.Such detectable labeling can involve radiolabeling, enzymatic labeling,or labeling with MRI paramagnetic chelates or microparticles orsuperparamagnetic particles; incorporation into ultrasound bubbles,microparticles, microspheres, emulsions, or liposomes; or conjugationwith optical dyes.

In another aspect of the present invention, methods for isolating KDR orKDR-expressing cells using the present binding polypeptides areprovided.

Additionally, the KDR and VEGF/KDR complex binding polypeptides of theinvention can be used as therapeutic agents, either as the solebioactive agent in a pharmaceutically acceptable composition orconjugated to (or in combination with) other therapeutic agents to treatdiseases or conditions involving KDR or VEGF/KDR complex, angiogenesisor diseases associated with a number of pathogens, including, forexample, malaria, HIV, SIV, Simian hemorrhagic fever, etc.

When the binding peptides disclosed herein are used as therapeuticagents, it may be advantageous to enhance the serum residence time ofthe peptides. This can be accomplished by: a) conjugating to the peptidea moiety, such as maleimide, that reacts with free sulfhydryl groups onserum proteins, such as serum albumin, b) conjugating to the peptide amoiety, such as a fatty acid, that binds non-covalently to serumproteins, especially serum albumin, c) conjugating to the peptide apolymer, such as PEG, that is known to enhance serum residence time,and/or d) fusing DNA that encodes the KDR-binding peptide to DNA thatencodes a serum protein such as human serum albumin or an antibody andexpressing the encoded fusion protein.

In another aspect of the invention, methods of screening polypeptidesidentified by phage display for their ability to bind to cellsexpressing the target are provided. These methods permit rapid screeningof the binding ability of polypeptides, including polypeptides withmonomeric affinities that are too low for evaluation in standardcell-binding assays. Additionally, these methods may be used to rapidlyassess the stability of the peptides in the presence of serum.

In another embodiment of the invention, a multimeric polypeptideconstruct having the ability to bind to KDR or VEGF/KDR complexcomprising at least one amino acid sequence selected from any of thepolypeptides described above is envisioned. In a particular embodiment,the polypeptide comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NOS: 20-86, 87-136, 187-192, 193-203, 207-259 and505-516. I a particular embodiment, the amino acid sequence selectedfrom the group consisting of: SEQ ID NOS: 137-186.

In one embodiment, the amino acid sequence further comprises N-terminaland/or C-terminal flanking peptides of one or more amino acids. Inanother embodiment, the amino acid sequence comprises a modificationselected from the group consisting of: an amino acid substitution, andamide bond substitution, a D-amino acid substitution, a glycosylatedamino acid, a disulfide mimetic substitution, an amino acidtranslocation, a retroinverso peptide, a peptoid, a retro-inversopeptoid, and a synthetic peptide.

In another embodiment, the polypeptide can be conjugated to a detectablelabel or a therapeutic agent, optionally further comprising a linker orspacer between the polypeptide and the detectable label or thetherapeutic agent.

In a particular embodiment, the detectable label or the therapeuticagent is selected from the group consisting of: an enzyme, a fluorescentcompound, a liposome, an optical dye, a paramagnetic metal ion, asuperparamagnetic particle, an ultrasound contrast agent and aradionuclide. In one embodiment, the therapeutic agent or detectablelabel comprises a radionuclide, including, for example, ¹⁸F, ¹²⁴I, ¹²⁵I,¹³¹I, ¹²³I, ⁷⁷Br, ⁷⁶Br, ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm,¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy,⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi,²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au or ¹⁹⁹Au. In aparticular embodiment, the therapeutic agent or detectable label furthercomprises a chelator, such as, for example, a compound selected from thegroup consisting of: formula 20, 21, 22, 23a, 23b, 24a, 24b, and 25. Ina particular embodiment, the detectable label comprises an ultrasoundcontrast agent that can comprise, for example, a phospholipid stabilizedmicrobubble or a microballoon comprising a gas. Alternatively, thedetectable label can comprise one or more paramagnetic metal ions or asuperparamagnetic particle and one or more chelators.

In another embodiment, the invention is directed to an ultrasoundcontrast agent comprising at least one KDR or VEGF/KDR complex bindingpolypeptide comprising an amino acid sequence of one of the followingand optionally further comprising N-terminal and/or C-terminal flankingpeptides of one or more amino acids described herein. In a particularembodiment, the gas filled microvesicles comprise phospholipidstabilized microbubbles or microballoons. In one embodiment, thephospholipid stabilized microbubbles or microballoons further comprise afluorinated gas.

In another embodiment, the invention is directed to a scintigraphicimaging agent comprising at least one KDR or VEGF/KDR complex bindingpolypeptide comprising an amino acid sequence of one of the followingand optionally further comprising N-terminal and/or C-terminal flankingpeptides of one or more amino acids described herein. In a particularembodiment, the scintigraphic imaging agent can comprise at least oneradionuclide useful in scintigraphic imaging and at least one KDR orVEGF/KDR complex binding moiety comprising a polypeptide of theinvention. In a particular embodiment, the scintigraphic imaging agentcan comprise at least one chelator selected from the group consistingof: formula 20, 21, 22, 23a, 23b, 24a, 24b and 25. In one embodiment,the radionuclide is selected from the group consisting of ^(99m)Tc and¹¹¹In.

In another embodiment, the invention is directed to an agent useful inradiotherapy comprising at least one KDR or VEGF/KDR complex bindingpolypeptide comprising an amino acid sequence of one of the followingand optionally further comprising N-terminal and/or C-terminal flankingpeptides of one or more amino acids described herein.

In another embodiment, the nvention is directed to an agent useful inradiotherapy comprising at least one radionuclide useful in radiotherapyand at least one KDR or VEGF/KDR complex binding moiety comprising apolypeptide of the invention. In a particular embodiment, the agent cancomprise at least one chelator selected from the group consisting of:formula 20, 21, 22, 23a, 23b, 24a, 24b and 25. In a particularembodiment, the radionuclide is selected from the group consisting of:¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm and ¹⁶⁶Ho.

In another embodiment, the invention is directed to a method ofsynthesizing a polypeptide or a multimeric polypeptide construct havingthe ability to bind KDR or VEGF/KDR complex comprising a cyclicpolypeptide formed by introducing an amide bond between two side chains.

In another embodiment, the invention is directed to a method ofsynthesizing a polypeptide or a multimeric polypeptide construct havingthe ability to bind KDR or VEGF/KDR complex comprising a polypeptide anda linker comprising at least one glycosylated amino acid selected fromthe group consisting or serine, threonine and homoserine.

In another embodiment, the invention is directed to a method ofsynthesizing a multimeric polypeptide construct having the ability tobind KDR or VEGF/KDR complex selected from the group consisting of D1,D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17,D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30 and D31,comprising: a) treating a purified peptide monomer with glutaric acidbis-N-hydroxysuccinimidyl ester; and b) contacting the peptide monomerin (a) with a second peptide monomer in the presence ofN,N-(Diisopropyl)aminomethylpolystyrene, thereby forming the multimericpolypeptide.

In another embodiment, the invention is directed to a multimericpolypeptide having the ability to bind to KDR or VEGF/KDR complexselected from the group consisting of: D1, D2, D3, D4, D5, D6, D7, D8,D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22,D23, D24, D25, D26, D27, D28, D29, D30 and D31.

In another embodiment, the invention is directed to a dimericpolypeptide construct having the ability to bind to KDR or VEGF/KDR,wherein each peptide of the dimer comprises a sequence of a polypeptideof the invention. In a particular embodiment, the amino acid sequence ofthe polypeptide is selected from the group consisting of: SEQ ID NOS:20-86, 87-136, 187-192, 193-203, 207-259 and 505-516. In a particularembodiment, the amino acid sequence of the polypeptide is selected fromthe group consisting of: SEQ ID NOS: 137-186.

Any of the dimmers of the invention can comprise N-terminal and/orC-terminal flanking peptides of one or more amino acids, as well as amodification such as, for example, an amino acid substitution, and amidebond substitution, a D-amino acid substitution, a glycosylated aminoacid, a disulfide mimetic substitution, an amino acid translocation, aretroinverso peptide, a peptoid, a retro-inverso peptoid or a syntheticpeptide. The dimeric constructs of the invention can be conjugated to adetectable label or a therapeutic agent, optionally further comprising alinker or spacer between the polypeptide and the detectable label or thetherapeutic agent. The detectable label or the therapeutic agent can be,for example, an enzyme, a fluorescent compound, a liposome, an opticaldye, one or more paramagnetic metal ions or a superparamagneticparticle, an ultrasound contrast agent or one or more radionuclides. Ina particular embodiment, the therapeutic agent or detectable labelcomprises one or more radionuclides. In a particular embodiment, adimeric construct can be labeled with one or more radionuclides such as,for example, ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, ⁷⁶Br, ^(99m)Tc, ⁵¹Cr,⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰La,⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re,¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹⁷mSn, ¹⁴⁹Pm,¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au or ¹⁹⁹ Au. In a particular embodiment, each peptideof the dimer is selected from an amino acid sequence selected from thegroup consisting of the sequences listed in Tables 1-11 and 27.

In another embodiment, the invention is directed to a multimericpolypeptide having the ability to bind to KDR or VEGF/KDR complex,wherein the multimeric polypeptide comprises at least one peptidemonomer comprising an amino acid sequence selected from the groupconsisting of those sequences listed in Tables 1-11 and 27.

In another embodiment, the invention is directed to a method ofinhibiting VEGF-induced vascular permeability comprising administeringand agent comprising a peptide of the invention. In a particularembodiment, the agent comprises D10.

In yet another embodiment, the present invention includes methods fordetecting, monitoring and/or evaluating a therapeutic response followingadministration of a contrast agent conjugated to KDR binding moietiesdescribed herein, including imaging before and after treatment. In apreferred embodiment imaging occurs before treatment and at one or moretimepoints after treatment. In one embodiment treatment is for acondition associated with angiogenesis and the treatment comprisesadministration of an anti-angiogenic agent. In another embodimenttreatment is for cancer or another hyperproliferative discease,particularly for KDR-expressing cancers such as prostate cancer. In oneembodiment the treatment for cancer comprises administration of ananti-cancer agent. In another embodiment, the treatment of cancercomprises radiotherapy, RF ablation or focused ultrasound treatment. Ina particular embodiment, the contrast agent is an ultrasound contrastagent. In a preferred embodiment the contrast agent comprises D5.

The present invention provides targeting vector-phospholipid conjugatesand particularly targeting peptide-phospholipid conjugates which areuseful in the preparation of gas filled ultrasound contrast agents. In apreferred embodiment the targeting peptide-phospholipid conjugatesinclude targeting peptides which exhibit high KDR binding affinity andthus are useful components of contrast agents for imaging and monitoringprocesses involving angiogenesis.

The present invention also provides monomeric and dimeric peptidephospholipid conjugates (also referred to herein as lipopeptides) whichare useful in preparing gas filled ultrasound contrast agents, andparticularly in preparing ultrasound contrast agents which target KDRand may be used for imaging and monitoring processes involvingangiogenesis.

The present invention also provides methods and processes for the largescale production of highly pure monomeric and dimeric peptidephospholipid conjugates, particularly monomeric and dimeric peptidephospholipids conjugates having high KDR binding affinity.

The present invention also provides methods and processes for the largescale production of highly pure dimeric peptide phospholipid conjugateshaving minimal levels of trifluoroacetic acid (TFA).

The present invention also provides methods for synthesizing monomericpeptides in high purity and the construction of peptide phospholipidconjuages from multiple peptide sub-units.

The present invention also provides monomeric peptides which bind KDR orthe VEGF/KDR complex with high affinity, as well as methods ofsynthesizing and using such monomeric peptides.

The present invention also provides targeted ultrasound contrast agentsprepared from such targeting vector-phospholipid conjugates. Suchtargeted ultrasound contrast agents are useful for imagingtarget-bearing tissue. In a preferred embodiment, the targetedultrasound contrast agents are targeted microbubbles and the targetingvector-phospholipid conjugates include targeting peptides which exhibithigh KDR binding affinity and thus are useful components of contrastagents for imaging KDR-bearing tissue and particularly for imaging andmonitoring of tumors and angiogenesis processes. Methods of preparingand using such targeted ultrasound contrast agents are also provided.

These and other aspects of the present invention will become apparentwith reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs illustrating the saturation binding curves ofbinding peptide/neutravidin-HRP complexes. FIG. 1A illustrates thesaturation binding curve for SEQ ID NO: 264 and SEQ ID NO: 294. FIG. 1Billustrates the saturation binding curve for SEQ ID NO: 277 and SEQ IDNO: 356. All peptides had a C-terminal biotin and JJ spacer.

FIG. 2 is a graph illustrating the binding of peptide/neutravidin-HRPcomplexes: control (biotinylated with spacer, and SEQ ID NOS: 264, 294,277 and 356) to KDR-transfected and Mock-transfected 293H cells at asingle concentration (5.55 nM). All peptides had a C-terminal biotin andJJ spacer.

FIG. 3 illustrates peptide structures, with and without both spacer(di(8-amino-3,6-dioxaoctanoic acid) “JJ”) and biotin tested in Example5((a) biotinylated SEQ ID NO: 264 with a JJ spacer; (b) SEQ ID NO: 264with an N-terminal biotin; (c) biotinylated SEQ ID NO: 294 with the JJspacer (d) biotinylated SEQ ID NO: 294).

FIG. 4 is a bar graph illustrating binding of peptide/neutravidin HRPcomplexes to KDR-transfected and mock-transfected 293H cells at single aconcentration (2.78 nM); peptides include (a) control (with spacer); (b)control; (c) biotinylated SEQ ID NO: 264 with a JJ spacer; (d) SEQ IDNO: 264 with an N-terminal biotin; and (e) biotinylated SEQ ID NO: 294with the JJ spacer; and (f) biotinylated SEQ ID NO: 294.

FIG. 5 is a bar graph illustrating specific binding (binding to KDRtransfected cells minus binding to Mock transfected cells) ofpeptide/neutravidin-HRP complexes with and without 40% rat serum. (a)SEQ ID NO: 294; (b) SEQ ID NO: 264; (c) SEQ ID NO: 277; (d) SEQ ID NO:356. Concentration of peptide/avidin HRP solutions was 6.66 nM for (a)and (b), 3.33 nM for (c), and 2.22 nM for (d). All peptides had aC-terminal biotin and JJ spacer.

FIG. 6 is a bar graph illustrating binding of polypeptide/avidin-HRPsolutions (SEQ ID NO: 294 and/or SEQ ID NO: 264) to mock- andKDR-transfected cells plotted as absorbance at 450 nm. The proportionsof control and KDR binding peptides used to form each tetrameric complexare indicated in the legend for each tested multimer.

FIG. 7 is a bar graph illustrating specific binding of a peptidecomprising SEQ ID NO: 294, and a biotinylated SEQ ID NO: 264 with a JJspacer/avidin-HRP complex to KDR transfected cells (background bindingto mock-transfected cells subtracted), plotted as absorbance at 450 nm.Increasing concentrations (as indicated in the X axis) of uncomplexedpeptides were added to the assay as indicated. Free SEQ ID NO: 264 wasable to decrease the binding of the SEQ ID NO: 264 complex toKDR-transfected cells.

FIG. 8 illustrates structures of binding polypeptide sequences tested inExample 6: SEQ ID NOS: 294, 368, 369, 337, 371 and 372.

FIG. 9 is a bar graph illustrating the binding of fluorescent beads toKDR-transfected and mock-transfected cells. Neutravidin-coated beadswith the indicated ligands attached were tested for binding toKDR-expressing and non-expressing 293H cells.

FIG. 10 is a bar graph illustrating percent inhibition of ¹²⁵I-labeledVEGF binding by binding polypeptides (a) acetylated SEQ ID NO: 294(without the modified C-terminus, GDSRVCWEDSWGGEVCFRYDP; SEQ ID NO:374); (b) SEQ ID NO: 263 (without the modified C-terminus,AGDSWCSTEYTYCEMIGT; SEQ ID NO: 375); (c) biotinylated SEQ ID NO: 264with a JJ spacer; and (d) SEQ ID NO: 277 (biotinylated with the JJspacer), at two concentrations (30 μM and 0.3 μM), to KDR-expressing293H transfectants.

FIG. 11 depicts chemiluminescent detection on film demonstrating thatactivated (phosphorylated) KDR was not detected in immunoprecipitatesfrom unstimulated (−V) HUVECs, but was abundant in immunoprecipitatesfrom VEGF-stimulated (+V) HUVECs (upper panel). Reprobing the blot withanti-KDR demonstrated that comparable amounts of total KDR were presentin both immunoprecipitates (lower panel).

FIG. 12 depicts chemiluminescent detection on film demonstrating theability of an anti-KDR antibody (1 μg/mL; indicated as “α-KDR”) topartially block VEGF-mediated phosphorylation.

FIG. 13 depicts chemiluminescent detection on film demonstrating theability of a KDR-binding polypeptide SEQ ID NO: 306 (10 μM) to blockVEGF-mediated KDR phosphorylation.

FIG. 14 is a bar graph showing binding of a Tc-labeled polypeptide (SEQID NO: 339) to KDR-transfected 293H cells.

FIG. 15 is a graph showing the percentage inhibition of ¹²⁵I-labeledVEGF binding by SEQ ID NO: 277, D2, D1, D3, and AQDWYYDEILSMADQLRHAFLSGG(SEQ ID NO: 376) at three different concentrations (10 μM, 0.3 μM, and0.03 μM) to KDR-transfected 293H cells. The results are from oneexperiment carried out in triplicate+/−S.D.

FIG. 16 is a photograph showing the ability of D1 to completely blockthe VEGF-induced phosphorylation of KDR in HUVECs at 10 nM and themajority of phosphorylation at 1 nM. Reprobing the blot for total KDR(lower panel) demonstrated that the effects of the tested compounds wasnot due to reduced sample loading. Homodimers (D2 and D3) composed ofthe two binding sequences contained in D1 did not interfere with thephosphorylation at up to 100 nM.

FIG. 17 is a graph showing that D1 potently blocks themigration/invasion of endothelial cells induced by VEGF. Migrating cellswere quantitated by fluorescence measurement after staining the migratedcells with a fluorescent dye.

FIG. 18 is a graph showing the binding of ¹²⁵I-labeled D5 to mock andKDR transfected 293H cells in the absence and presence of 40% mouseserum.

FIG. 19 is a graph showing the specific binding (KDR-MOCK) of¹²⁵I-labeled D5 to KDR-transfected 293H cells in the absence andpresence of 40% mouse serum.

FIG. 20 is a graph of plasma clearance as percent injected dose per mLversus time.

FIG. 21 shows SE-HPLC profiles of plasma from the Superdex peptidecolumn. Top panel, sample injected; followed by 0 min, 30 min, and 90min. The insert within each panel shows time point, animal number andvolume injected for HPLC analysis.

FIG. 22 is a graph showing the results of testing of KDR peptides inHUVEC proliferation assay. A: D6; B: SEQ ID NO: 277; C: SEQ ID NO: 377(AEGTGDLHCYFPWVCSLDPGPEGGGK; negative control); F: SEQ ID NO: 377;negative control.

FIGS. 23A and 23B show the kinetic analysis of D1 (see FIG. 36), bindingto murine KDR-Fc. All sensograms are fit to the bivalent analyte model.

FIGS. 24A and 24B show the kinetic analysis of D7, a heterodimer of SEQID NO: 264 and SEQ ID NO: 294. All sensograms are fit to the bivalentanalyte model.

FIGS. 25A and 25B show the kinetic analysis of fluorescein labeled SEQID NO: 277 binding to murine KDR-Fc. All sensograms are fit to the 1:1Langmuir model.

FIG. 26 depicts examples of alpha, beta, gamma or delta dipeptide orturn mimics (such as α, β, γ, or δ turn mimics), shown in panels 1, 2and 3.

FIG. 27 shows an oxime linker. The amino acids containing anaminoalcohol function (4), and containing an alkoxyamino function (5),are incorporated into the peptide chain, not necessarily at the end ofthe peptide chain.

FIG. 28 shows an example of cyclization of cysteine with a pendantbromoacetamide function.

FIG. 29 is a schematic showing the formation of cyclic peptides with athiazolidine linkage via intramolecular reaction of peptide aldehydeswith cysteine moieties.

FIG. 30 is a schematic showing lactam surrogate for the disulfide bondvia quasiorthogonal deprotection of Lys and Asp followed by on-resincyclization and cleavage from resin.

FIG. 31 is a schematic showing lactam surrogate for the disulfide bondvia quasiorthogonal deprotection of Lys and Asp using allyl-basedprotecting groups followed by on-resin cyclization and cleavage fromresin.

FIG. 32 is a schematic depicting Grubbs Olefin Metathesis Cyclization.

FIG. 33 shows phospholipid structures.

FIGS. 34A-F depict preferred structures of chelators.

FIG. 35 shows the structure of a chelating agent.

FIG. 36 shows dimer 1 (D1; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277)[(Biotin-JJK-(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK(SEQID NO: 337))-NH)CONH₂]—NH₂).

FIG. 37 shows dimer 2 (D2; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277)[(Biotin-JJK-(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-AGPTWCEDDWYYCWLFGTJK(SEQID NO: 493))-NH)CONH₂]—NH₂)_(.)

FIG. 38 shows dimer 3 (D3; Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)[(Biotin-JJK-(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK(SEQID NO: 337))-NH)CONH₂]—NH₂).

FIG. 39 shows dimer 4 (D4; Ac-AGPTWCEDDWYYCWLFGTJK(SEQ ID NO:338)[DOTA-JJK-(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK(SEQID NO: 337))-NH)CONH₂]—NH₂).

FIG. 40 shows dimer 5 (D5; Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)(JJ-C(═O)(CH₂)₃C(═O)—K—NH(CH₂)₄—(S)—CH((Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ IDNO: 277))-NH)CONH₂)—NH₂).

FIG. 41 shows dimer 8 (D8; Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ ID NO:356) {Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ ID NO:356)(J-Glut-)-NH₂}K(Biotin-JJ)-NH₂).

FIG. 42 shows dimer 9 (D9; Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ ID NO:356){[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294)(JJ-Glut-)]-NH₂}K—NH₂).

FIG. 43 shows dimer 10 (D10Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294)(JJ-Glut-NH(CH₂)₄—(S)—CH(PnAO6-Glut-NH)(C═O—)]—NH₂}—NH₂).

FIG. 44 shows dimer 11 (D11; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277){Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)[JJ-Glut-NH(CH₂)₄—(S)—CH(DOTA-JJ-NH—)(C═O)—]—NH₂}—NH₂).

FIG. 45 shows dimer 12 (D12; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){[PnAO6-Glut-K(Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)(—C(═O)CH₂(OCH₂CH₂)₂OCH₂C(═O)—)—NH₂]}—NH₂).

FIG. 46 shows dimer 13 (D13; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)[JJ-Glut-K(BOA)]—NH₂}—NH₂).

FIG. 47 shows dimer 14 (D14; Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ ID NO:356){PnAO6-Glut-K[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:477)(JJ-Glut)-NH₂]}—NH₂).

FIG. 48 shows dimer 15 (D15; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294)[JJ-Glut]-NH₂]-K(PnAO6-Glut)}—NH₂).

FIG. 49 shows dimer 16 (D16; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){PnAO6-Glut-K [Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294)[—C(═O)CH₂—O—(CH₂CH₂O)₂CH₂C(═O)NH(CH₂)₃—O—(CH₂CH₂O)₂(CH₂)₃NHC(═O)CH₂—O—(CH₂CH₂O)₂CH₂C(═O)—]—NH₂]}—NH₂).

FIG. 50 shows dimer 17 (D17; Ac-AQDWYYDEILJGRGGRGGRGGK(SEQ ID NO: 478){K[Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)(JJ-Glut)-NH₂]}—NH₂).

FIG. 51 shows dimer 18 (D18; Ac-APGTWCDYDWEYCWLGTFGGGK(SEQ ID NO: 497){PnAO6-Glut-K[Ac-GVDFRCEWSDWGEVGCRSPDYGGGK(SEQ ID NO:489)(JJ-Glut)-NH₂]}—NH₂).

FIG. 52 shows dimer 19 (D19; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){Biotin-K[Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)(JJ-Glut)-NH₂]}-NH₂).

FIG. 53 shows dimer 20 (D20; (((-JJ)-AGPTWCEDDWYYCWLFGTGGGGK(SEQ ID NO:480)-NH₂)-Glut-JJ)VCWEDSWGGEVCFRYDPGGG(SEQ ID NO: 370)-NH₂).

FIG. 54 shows dimer 21 (D21; [(-JJ)-AGPTWCEDDWYYCWLFGTGGGGK(SEQ ID NO:480)(PnAO6-Glut)-NH₂]-Glut-(JJ)-VCWEDSWGGEVCFRYDPGGG(SEQ ID NO:370)-NH₂).

FIG. 55 shows dimer 22 (D22; Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294) {JJ-Glut-JJ-AGPTWCEDDWYYCWLFTGGGK(SEQ ID NO: 481)-NH₂}—NH₂).

FIG. 56 shows dimer 23 (D23; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)[JJ-Glut-K(SATA)]—NH₂}—NH₂. D23 is dimer D5 functionalized with the SATA(S-Acetylthioacetyl) group).

FIG. 57 shows dimer 24 (D24; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){SATA-JJK[Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)(JJ-Glut)-NH₂]}—NH₂).

FIG. 58 shows dimer 25 (D25; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294)[JJ-Glut-NH(CH₂)₄—(S)—CH(NH₂)C(═O)-]-NH₂}—NH₂).

FIG. 59 shows dimer 26 (D26; AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){(-Glut-JJ-VCWEDSWGGEVCFRYDPGGG(SEQ ID NO: 370)-NH₂)—K}—NH₂).

FIG. 60 shows dimer 27 (D27; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277){Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)[S(GalNAc-alpha-D)-G-S(GalNAc-alpha-D)-Glut-S(GalNAc-alpha-D)-G-S(GalNAc-alpha-D)-NH(CH₂)₄—(S)—CH(Biotin-JJNH—)C(═O)—]—NH₂}—NH₂).

FIG. 61 shows dimer 28 (D28; comprising AQEPEGYAYWEVITLYHEEDGDGGK (SEQID NO: 305) and AQAFPRFGGDDYWIQQYLRYTDGGK (SEQ ID NO: 306)).

FIG. 62 shows dimer 29 (D29; comprising AGPTWCEDDWYYCWLFGTGGGK (SEQ IDNO: 277) and VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 337)).

FIG. 63 shows dimer 6 (D6; comprising GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ IDNO: 294) and AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO: 277)).

FIG. 64 shows dimer 7 (D7; comprising GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ IDNO: 294) and AGPKWCEEDWYYCMITGTGGGK (SEQ ID NO: 264)).

FIG. 65 is a graph showing the inhibition of tumor growth by D6 as afunction of D6 concentration.

FIG. 66 shows that D27 (squares) with its glycosylation and modifiedspacer is able to block the effects of VEGF in the migration assay toblock VEGF-stimulated migration even more potently than D25 (diamonds),which lacks those chemical modifications.

FIGS. 67A and 67B show that Adjunct A enhances the potency of D6 inblocking the biological effects of VEGF in a migration assay withcultured HUVECs. FIG. 67A: Diamonds: D6 alone at the indicatedconcentrations. Squares: D6 at the indicated concentrations plus 100 nMAdjunct A (constant). FIG. 67B shows the structure of Adjunct A.

FIG. 68 is a schematic showing Scheme 1 (synthesis of Peptide 2).

FIG. 69 is a schematic showing Scheme 2 (synthesis of Peptide 4).

FIG. 70 is a schematic showing Scheme 3 (synthesis of D27).

FIG. 71 depicts % inhibition±s.d. of specific ¹²⁵I-VEGF binding toKDR-transfected cells by SEQ ID NO: 504 (squares) and D1 (diamonds).

FIG. 72 depicts % maximum VEGF-stimulated migration±s.d. of HUVEC cellsin the presence of the indicated concentrations of SEQ ID NO: 504(diamonds) or D1 (squares).

FIG. 73 is a graphical representation showing total binding of complexesof control peptide and the test peptides (SEQ ID NOS: 321, 320 and 323)with ¹²⁵I-streptavidin (in the presence of VEGF) to mock-transfected andKDR-transfected cells. Only the complex containing SEQ ID NO: 321 showedspecific binding (KDR-mock).

FIG. 74 is a graphical representation showing specific binding ofcomplexes of peptide (SEQ ID NO: 321) and ¹²⁵I-streptavidin (in theabsence and presence of VEGF) to KDR-transfected cells at variousconcentrations (0-13.33 nM) of peptide-¹²⁵I-streptavidin complex.

FIG. 75 shows that homodimeric D8 (squares) does not block the effectsof VEGF in the migration assay as carried out in Example 28 as well theheterodimeric D17 (diamonds).

FIG. 76 is a schematic showing the synthesis of cyclic lactam peptides(sample procedure).

FIG. 77 is a graphical representation showing binding of SEQ ID NO: 482derivatives with different spacer length and biotin. Derivatives havenone, one J and two J spacers, respectively, in between the SEQ ID NO:482 targeting sequence and biotin.

FIG. 78 depicts the binding of Tc-labeled D10 to KDR-transfected 293Hcells as described in Example 32. Mock=mock-transfected.Trans=KDR-transfected. MS=mouse serum.

FIGS. 79A-G show derivatives of binding peptides of the invention.

FIG. 80 summarizes the results of a radiotherapy study with D13conducted in nude mice implanted with PC3 tumors. Each plotted linerepresents the growth over time for an individual tumor in a treatedmouse, except for the heavy dashed line, which represents the averagetumor growth in a set of untreated mice, as described in Example 34.

FIG. 81 shows uptake and retention of bubble contrast in the tumor up to30 minutes post injection for suspensions of microbubbles conjugated toSEQ ID NO: 356. In contrast, the same bubbles showed only transient (nomore than 10 minutes) visualization/bubble contrast in the AOI situatedoutside the matrigel or tumor site (see FIGS. 82 and 83).

FIG. 82 shows uptake and retention of bubble contrast in the tumor up to30 minutes post injection for suspensions of microbubbles conjugated toa SATA-modified peptide comprising SEQ ID NO: 356. In contrast, the samebubbles showed only transient (no more than 10 minutes)visualization/bubble contrast in the AOI situated outside the matrigel.

FIG. 83 shows uptake and retention of bubble contrast in the matrigel upto 30 minutes post injection for suspensions of microbubbles conjugatedto a SATA-modified peptide comprising SEQ ID NO: 294. In contrast, thesame bubbles showed only transient (no more than 10 minutes)visualization/bubble contrast in the AOI situated outside the matrigel.

FIG. 84 is a graph showing the results of in vitro binding assays.Microvascular endothelial cells (MVECs, Cascade Biologics, Portland,Oreg.) were used to assess the in vitro efficacy of D6 and relatedanalogues for their ability to inhibit VEGF-stimulated proliferation.

FIG. 85 shows a typical example of peptide-conjugated ultrasoundcontrast agents bound to KDR-or mock-transfected cells in presence of10% human serum (magnification: 100×).

FIG. 86 is a schematic representation of the synthesis scheme used toprepare4-{2-(2-Hydroxyimino-1,1-dimethylpropylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyricacid, N-hydroxysuccinimide ester (Compound B) using4-{2-(2-Hydroxyimino-1,1-dimethyl-propylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyricacid as a starting reagent.

FIGS. 87A-C are schematic representations depicting synthesis schemesand structures for Dimer D30. FIG. 87A shows the synthesis scheme forthe preparation of Compound 3.

FIG. 87B shows the synthesis scheme for dimer D30: Preparation ofAc-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337){[PnAO6-Glut-K(-Glut-B—NH(CH₂)₄—(S)—CH(Ac-AQDWYYDEILJGRGGRGGRGG(SEQID NO: 478)-NH)C(═O)NH₂]—NH₂}—NH₂: D30 from Compound 3 and Compound 4.FIG. 87C shows the structure of dimer D30.

FIGS. 88A-D are schematic representations depicting synthesis schemesand structures for dimer D31. FIG. 88A shows the synthesis scheme forthe preparation of Compound 2.

FIG. 88B shows the synthesis scheme for the preparation of Compound 4 (apeptide related to SEQ ID NO: 374). FIG. 88C depicts the synthesisscheme and structure for dimer D31 (i.e., Preparation ofAc-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277)[Ac-VCWEDSWGGEVCFRYDPGGGK(SEQID NO: 337)[SGS-Glut-SGS-(S)—NH(CH₂)₄—CH(Biotin-B—NH)—C(═O)]—NH₂]—NH₂).FIG. 88D shows the structure of D31.

FIG. 89 is a graph that shows competition of targeted bubbles bycorresponding free peptide.

FIG. 90 is a graph that shows competition of targeted bubbles conjugateto D23 by corresponding free peptide.

FIG. 91 is a graph that shows competition of targeted bubbles with freedimer.

FIG. 92 is a graph showing binding values obtained with the suspensionsof microbubbles conjugated to the D23, SATA-modified SEQ ID NO: 480,SATA-modified SEQ ID NO: 294 or SEQ ID NO: 294/SEQ ID NO: 480 (50/50)are equivalent (see Example 43).

FIG. 93 is a graph showing dimer D10 blocks the increased peritonealvascular permeability induced by VEGF injected intraperitoneally.Solutions containing the indicated additions were injectedintraperitoneally, and their effect on peritoneal permeability wasassessed by measuring the O.D. of the injected fluid at 590 nm afteradministering Evan's Blue dye i.v. as described in Example 44.

FIG. 94 is a graph showing uptake and retention of bubble contrast inthe tumor up to 30 minutes post injection for suspensions ofphospholipid stabilized microbubbles conjugated to a heteromultimericconstruct (D23).

FIG. 95 is a graph depicting the binding of Lu-D13 to KDR-transfected293H cells. Mock=mock-transfected. Trans=KDR-transfected. MS=mouseserum.

FIG. 96 is a graph showing the specific binding of a Tc-labeledpolypeptide (SEQ ID NO: 339) to KDR-transfected 293H cells aftersubtracting the binding to mock-transfected 293H cells.

FIG. 97 is a bar graph demonstrating that Tc-labeled SEQ ID NO: 277 withTc-chelate binding to KDR-transfected 293H cells is inhibited by about80% in the presence of 40% rat serum.

FIG. 98 is a graph showing the ability of a contrast agent comprising D5to detect early anti-angiogenic therapeutic effect of treatment with atherapeutic agent. Imax, LPO and area of a tumor in an OFA rat areshown.

FIG. 99 is a graph showing the ability of a contrast agent comprising D5to detect early anti-angiogenic therapeutic effect of treatment with atherapeutic agent. Imax, LPO and area of a tumor in a second rat areshown.

FIG. 100 is a graph showing the ability of a contrast agent comprisingD5 to detect early anti-angiogenic therapeutic effect of treatment witha therapeutic agent. Imax, LPO and area of a tumor in a third rat areshown.

FIG. 101 is a graph showing the Imax, LPO and area of a tumor in afourth rat which did not receive a therapeutic agent.

FIG. 102 is a graph showing variation in echographic parameters 48 hoursafter sunitinib treatment in the rats referenced in FIGS. 98, 99 and100.

FIG. 103 provides immunohistochemistry images of a mammary tumor in therat referenced in FIG. 98.

FIG. 104 provides immunohistochemistry images of a mammary tumor in therat referenced in FIG. 101.

FIG. 105 illustrates a method for the production of a monomeric peptidephospholipid conjugate (1) from a linear peptide monomer (2).

FIG. 106 illustrates a monomeric peptide phospholipid conjugate (1)including a peptide with high binding affinity for KDR.

FIG. 107 illustrates a method for the production of a precursor dimerpeptide (16) from peptide monomers.

FIG. 108 illustrates a method for the conjugation of the precursor dimerpeptide shown in FIG. 105 to DSPE-PEG2000-NH₂ to form a dimeric peptidephospholipid conjugate (11) containing peptides which bind with highaffinity to KDR.

FIG. 109 illustrates a dimeric peptide-phospholipid conjugate (11)containing peptides which bind with high affinity to KDR.

FIG. 110 illustrates a method for the production of dimerpeptide-phospholipid conjugates (such as (21)) having minimal levels ofTFA.

FIG. 111 illustrates another method for the production of dimerpeptide-phospholipid conjugates (such as (21)) having minimal levels ofTFA.

FIG. 112 illustrates another method for the production of dimerpeptide-phospholipid conjugates having minimal levels of TFA.

FIG. 113 illustrates another representative monomeric peptide (32)having a high binding affinity for KDR.

FIG. 114 illustrates another monomeric peptide-phospholipid conjugate(31) which includes the monomeric peptide shown in FIG. 113.

FIGS. 115A-C show images obtained by using the dimerpeptide-phospholipid conjugate (11) (shown in FIG. 109) in a contrastagent at: 1) baseline (FIG. 115A); 2) after 25 minutes (FIG. 115B); and3) after subtraction of the baseline and free circulating bubbles (FIG.115C).

FIGS. 116A-C show images obtained by using the monomeric phospholipidpeptide conjugate (1) (shown in FIG. 106) in a contrast agent atbaseline (FIG. 116A); after 25 minutes (FIG. 116B); and aftersubtraction of the baseline and free circulating bubbles (FIG. 116C).

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

DEFINITIONS

In the following sections, the term “recombinant” is used to describenon-naturally altered or manipulated nucleic acids, host cellstransfected with exogenous nucleic acids, or polypeptides expressednon-naturally, through manipulation of isolated DNA and transformationof host cells. Recombinant is a term that specifically encompasses DNAmolecules that have been constructed in vitro using genetic engineeringtechniques, and use of the term “recombinant” as an adjective todescribe a molecule, construct, vector, cell, polypeptide orpolynucleotide specifically excludes naturally occurring such molecules,constructs, vectors, cells, polypeptides or polynucleotides.

The term “bacteriophage” is defined as a bacterial virus containing aDNA core and a protective shell built up by the aggregation of a numberof different protein molecules. The terms “bacteriophage” and “phage”are used herein interchangeably.

The term “polypeptide” is used to refer to a compound of two or moreamino acids joined through the main chain (as opposed to side chain) bya peptide amide bond (—C(:O)NH—). The term “peptide” is usedinterchangeably herein with “polypeptide” but is generally used to referto polypeptides having fewer than 40, and preferably fewer than 25 aminoacids.

The term “binding polypeptide” as used herein refers to any polypeptidecapable of forming a binding complex with another molecule. Anequivalent term sometimes used herein is “binding moiety”. “KDR bindingpolypeptide” is a polypeptide that forms a complex in vitro or in vivowith vascular endothelial growth factor receptor-2 (or KDR, Flk-1);“VEGF/KDR complex binding polypeptide” is a polypeptide that forms acomplex in vitro or in vivo with a binding complex formed betweenvascular endothelial growth factor (VEGF) and KDR, in particular thecomplex of homodimeric VEGF and one or two KDR molecules that isbelieved to form at the surface of endothelial cells duringangiogenesis. Specific examples of KDR and VEGF/KDR binding polypeptidesinclude but are not limited to the peptides presented in Tables 1-7,infra, and include hybrid and chimeric polypeptides incorporating suchpeptides. Also included within the definition of KDR and VEGF/KDRcomplex binding polypeptides are polypeptides that are modified oroptimized as disclosed herein.

Specific examples of such modifications are discussed in detail infra,but include substitution of amino acids for those in the parentpolypeptide sequence to optimize properties, obliterate an enzymecleavage site, etc.; C- or N-terminal amino acid substitutions orelongations, e.g., for the purpose of linking the binding polypeptide toa detectable imaging label or other substrate, examples of whichinclude, e.g., addition of a polyhistidine “tail” in order to assist inpurification; truncations; amide bond changes; translocations;retroinverso peptides; peptoids; retroinversopeptoids; the use ofN-terminal or C-terminal modifications or linkers, such as polyglycineor polylysine segments; alterations to include functional groups,notably hydrazide (—NH—NH₂) functionalities or the C-terminal linker-Gly-Gly-Gly-Lys (SEQ ID NO: 18), to assist in immobilization of bindingpeptides according to this invention on solid supports or for attachmentof fluorescent dyes; pharmacokinetic modifications, structuralmodifications to retain structural features, formation of salts toincrease water solubility or ease of formulation, and the like.

In addition to the detectable labels described further herein, othersuitable substrates for the binding polypeptides include a tumorcidalagent or enzyme, a liposome (e.g., loaded with a therapeutic agent, anultrasound appropriate gas, or both), or a solid support, well, plate,bead, tube, slide, filter or dish. Moreover, dimers or multimers of oneor more KDR or VEGF/KDR binding polypeptides can be formed. Suchconstructs may, for example, exhibit increased ability to bind to KDR.All such modified binding polypeptides are also considered KDR orVEGF/KDR complex binding polypeptides so long as they retain the abilityto bind the KDR or VEGF/KDR targets.

“Homologues” of the binding polypeptides described herein can beproduced using any of the modification or optimization techniquesdescribed herein or known to those skilled in the art. Such homologouspolypeptides will be understood to fall within the scope of the presentinvention and the definition of KDR and VEGF/KDR complex bindingpolypeptides so long as the substitution, addition, or deletion of aminoacids or other such modification does not eliminate its ability to bindeither KDR or VEGF/KDR complex. The term “homologous”, as used herein,refers to the degree of sequence similarity between two polymers (i.e.,polypeptide molecules or nucleic acid molecules). Where the samenucleotide or amino acid residue or one with substantially similarproperties (i.e., a conservative substitution) occupies a sequenceposition in the two polymers under comparison, then the polymers arehomologous at that position. For example, if the amino acid residues at60 of 100 amino acid positions in two polypeptide sequences match or arehomologous then the two sequences are 60% homologous.

The homology percentage figures referred to herein reflect the maximalhomology possible between the two polymers, i.e., the percent homologywhen the two polymers are so aligned as to have the greatest number ofmatched (homologous) positions. Polypeptide homologues within the scopeof the present invention will be at least 70% and preferably greaterthan 80% homologous to at least one of the KDR or VEGF/KDR bindingsequences disclosed herein.

The term “binding” refers to the determination by standard assays,including those described herein, that a binding polypeptide recognizesand binds reversibly to a given target. Such standard assays include,but are not limited to equilibrium dialysis, gel filtration, and themonitoring of spectroscopic changes that result from binding.

The term “specificity” refers to a binding polypeptide having a higherbinding affinity for one target over another. The term “KDR specificity”refers to a KDR binding moiety having a higher affinity for KDR than foran irrelevant target. The term “VEGF/KDR specificity” refers to aVEGF/KDR complex binding moiety having a higher affinity for a VEGF/KDRcomplex than for another given target. Binding specificity can becharacterized by a dissociation equilibrium constant (K_(D)) or anassociation equilibrium constant (K_(a)) for the two tested targetmaterials, or can be any measure of relative binding strength. Thebinding polypeptides according to the present invention are specific forKDR or VEGF/KDR complex and preferably have a K_(D) for KDR or VEGF/KDRcomplex that is lower than 10 μM, more preferably less than 1.0 μM, mostpreferably less than 0.5 μM or even lower.

The term “patient” as used herein refers to any mammal, especiallyhumans.

The term “pharmaceutically acceptable” carrier or excipient refers to anon-toxic carrier or excipient that can be administered to a patient,together with a compound of this invention, such that it does notdestroy the biological or pharmacological activity thereof. Thefollowing common abbreviations are used throughout this specification:9-fluorenylmethyloxycarbonyl (fmoc or Fmoc), 1-hydroxybenzotriazole(HOBt), N,N′-diisopropylcarbodiimide (DIC), acetic anhydride (Ac₂O),(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),trifluoroacetic acid (TFA), Reagent B(TFA:H₂O:phenol:triisopropylsilane, 88:5:5:2), N,N-diisopropylethylamine(DIEA), O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate(HATU), N-hydroxysuccinimide (NHS), solid phase peptide synthesis(SPPS), dimethyl sulfoxide (DMSO), dichloromethane (DCM),dimethylformamide (DMF), and N-methylpyrrolidinone (NMP).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel binding moieties that bind KDR or acomplex of VEGF and KDR. Such binding moieties make possible theefficient detection, imaging and localization of activated endothelialcells exhibiting upregulated KDR expression and binding to VEGF. Suchendothelial cells are characteristic of active angiogenesis, andtherefore the polypeptides described herein provide a means ofdetecting, monitoring and localizing sites of angiogenesis. Inparticular, the binding polypeptides of this invention, whenappropriately labeled, are useful for detecting, imaging and localizingtumor-induced angiogenesis. Thus, the binding polypeptides can be usedto form a variety of diagnostic and therapeutic agents for diagnosingand treating neoplastic tumor growth or other pathogenic instances ofangiogenesis.

In addition, the binding polypeptides can themselves be used astherapeutic agents. Specific KDR and VEGF/KDR complex bindingpolypeptides according to the present invention were isolated initiallyby screening of phage display libraries, that is, populations ofrecombinant bacteriophage transformed to express an exogenous peptide ontheir surface. In order to isolate new polypeptide binding moieties fora particular target, such as KDR or VEGF/KDR, screening of large peptidelibraries, for example using phage display techniques, is especiallyadvantageous, in that very large numbers (e.g., 5×10⁹) of potentialbinders can be tested and successful binders isolated in a short periodof time.

In order to prepare a phage library of displaying polypeptides to screenfor binding polypeptides such as KDR or VEGF/KDR complex bindingpolypeptides, a candidate binding domain is selected to serve as astructural template for the peptides to be displayed in the library. Thephage library is made up of a multiplicity of analogues of the parentaldomain or template. The binding domain template may be a naturallyoccurring or synthetic protein, or a region or domain of a protein. Thebinding domain template may be selected based on knowledge of a knowninteraction between the binding domain template and the binding target,but this is not critical. In fact, it is not essential that the domainselected to act as a template for the library have any affinity for thetarget at all: Its purpose is to provide a structure from which amultiplicity (library) of similarly structured polypeptides (analogues)can be generated, which multiplicity of analogues will hopefully includeone or more analogues that exhibit the desired binding properties (andany other properties screened for).

In selecting the parental binding domain or template on which to basethe variegated amino acid sequences of the library, the most importantconsideration is how the variegated peptide domains will be presented tothe target, i.e., in what conformation the peptide analogues will comeinto contact with the target. In phage display methodologies, forexample, the analogues will be generated by insertion of synthetic DNAencoding the analogues into phage, resulting in display of the analogueon the surfaces of the phage. Such libraries of phage, such as M13phage, displaying a wide variety of different polypeptides, can beprepared using techniques as described, e.g., in Kay et al., PhageDisplay of Peptides and Proteins: A Laboratory Manual (Academic Press,Inc., San Diego, 1996) and U.S. Pat. No. 5,223,409 (Ladner et al.),incorporated herein by reference.

In isolating the specific polypeptides according to this invention,seven cyclic peptide (or “loop”) libraries, designated TN6/VI, TN7/IV,TN8/IX, TN9/IV, TN10/IX, TN12/I, and MTN13/I, and a linear library,designated Lin20, were used. Each library was constructed for expressionof diversified polypeptides on M13 phage. The seven libraries having a“TN” designation were designed to display a short, variegated exogenouspeptide loop of 6, 7, 8, 9, 10, 12 or 13 amino acids, respectively, onthe surface of M13 phage, at the amino terminus of protein III. Thelibraries are designated TN6/VI (having a potential 3.3×10¹² amino acidsequence diversity), TN7/IV (having a potential 1.2×10¹⁴ amino acidsequence diversity), TN8/IX (having a potential 2.2×10¹⁵ amino acidsequence diversity), TN9/IV (having a potential 4.2×10¹⁶ amino acidsequence diversity), TN10/IX (having a potential 3.0×10¹⁶ amino acidsequence diversity), TN12/I (having a sequence diversity of 4.6×10¹⁹),MTN13/I (having a potential 8.0×10¹⁷ amino acid sequence diversity), andLin20 (having a potential 3.8×10²⁵ amino acid sequence diversity).

The TN6/VI library was constructed to display a single microproteinbinding loop contained in a 12-amino acid template. The TN6/VI libraryutilized a template sequence ofXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Cys-Xaa₁₀-Xaa₁₁-Xaa₁₂. The aminoacids at positions 2, 3, 5, 6, 7, 8, 10, and 11 of the template werevaried to permit any amino acid except cysteine (Cys). The amino acidsat positions 1 and 12 of the template were varied to permit any aminoacid except cysteine (Cys), glutamic acid (Glu), isoleucine (Ile),lysine (Lys), methionine (Met), and threonine (Thr).

The TN7/IV library was constructed to display a single microproteinbinding loop contained in a 13-amino acid template. The TN7/IV libraryutilized a template sequence ofXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Xaa₁₁-Xaa₁₂-Xaa₁₃. Theamino acids at amino acid positions 1, 2, 3, 5, 6, 7, 8, 9, 11, 12, and13 of the template were varied to permit any amino acid except cysteine(Cys).

The TN8/IX library was constructed to display a single microproteinbinding loop contained in a 14-amino acid template. The TN8/IX libraryutilized a template sequence ofXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Cys-Xaa₁₂-Xaa₁₃-Xaa₁₄.The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, and 14in the template were varied to permit any amino acid except cysteine(Cys).

The TN9/IV library was constructed to display a single microproteinbinding loop contained in a 15-amino acid template. The TN9/IV libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Cys-Xaa₁₃-Xaa₁₄-Xaa₁₅.The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and15 in the template were varied to permit any amino acid except cysteine(Cys).

The TN10/IX library was constructed to display a single microproteinbinding loop contained in a 16-amino acid template. The TN10/IX libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Cys-Xaa₁₄-Xaa₁₅-Xaa₁₆.The amino acids at positions 1, 2, 15, and 16 in the template werevaried to permit any amino acid selected from a group of 10 amino acids:D, F, H, L, N, P, R, S, W, or Y). The amino acids at positions 3 and 14in the template were varied to permit any amino acid selected from agroup of 14 amino acids: A, D, F, G, H, L, N, P, Q, R, S, V, W, or Y).The amino acids at positions 5, 6, 7, 8, 9, 10, 11, and 12 in thetemplate were varied to permit any amino acid except cysteine (Cys).

The TN12/I library was constructed to display a single microproteinbinding loop contained in an 18-amino acid template. The TN12/I libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Cys-Xaa₁₆-Xaa₁₇-Xaa₁₈.The amino acids at position 1, 2, 17, and 18 in the template were variedto permit any amino acid selected from a group of 12 amino acids: A, D,F, G, H, L, N, P, R, S, W, or Y). The amino acids at positions 3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, and 16 were varied to permit any amino acidexcept cysteine (Cys).

The MTN13/I library was constructed to display a single microproteinbinding loop contained in a 19-amino acid template featuring twovariable regions of equal size (i.e., eight amino acids) separated by aconstant region of three amino acids (Ser-Gly-Pro). The MTN13/I libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Ser-Gly-Pro-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Cys-Xaa₁₇-Xaa₁₈-Xaa₁₉(SEQ ID NO: 19). The amino acids at position 1, 2, 3, 5, 6, 7, 8, 12,13, 14, 15, 17, 18, and 19 in the template were varied to permit anyamino acid except cysteine (Cys).

The Lin20 library was constructed to display a single linear peptide ina 20-amino acid template. The amino acids at each position in thetemplate were varied to permit any amino acid except cysteine (Cys).

The binding polypeptides provided herein can include additions ortruncations in the N- and/or C-termini. Such modified bindingpolypeptides are expected to bind KDR or VEGF/KDR complex. For example,the -GGGK linker present at the N-terminus of some of the bindingpolypeptides provided herein is an optional linker. Therefore,polypeptides having the same sequence, except without the terminal -GGGKsequence, are also encompassed by the present invention. In addition,binding polypeptides comprising the loop portion of the templates andsequences provided herein are expected to bind KDR and/or VEGF/KDRcomplex and are also encompassed by the present invention. The loopportion of the templates and sequences includes the sequences betweenand including the two cysteine residues that are expected to form adisulfide bond, thereby generating a peptide loop structure.Furthermore, the binding polypeptides of the present invention caninclude additional amino acid residues at the N- and/or C-termini.

The phage display libraries were created by making a designed series ofmutations or variations within a coding sequence for the polypeptidetemplate, each mutant sequence encoding a peptide analogue correspondingin overall structure to the template except having one or more aminoacid variations in the sequence of the template. The novel variegated(mutated) DNA provides sequence diversity, and each transformant phagedisplays one variant of the initial template amino acid sequence encodedby the DNA, leading to a phage population (library) displaying a vastnumber of different but structurally related amino acid sequences. Theamino acid variations are expected to alter the binding properties ofthe binding peptide or domain without significantly altering itsstructure, at least for most substitutions. It is preferred that theamino acid positions that are selected for variation (variable aminoacid positions) will be surface amino acid positions, that is, positionsin the amino acid sequence of the domains that, when the domain is inits most stable conformation, appear on the outer surface of the domain(i.e., the surface exposed to solution). Most preferably the amino acidpositions to be varied will be adjacent or close together, so as tomaximize the effect of substitutions.

As indicated previously, the techniques discussed in Kay et al., PhageDisplay of Peptides and Proteins: A Laboratory Manual (Academic Press,Inc., San Diego, 1996) and U.S. Pat. No. 5,223,409 are particularlyuseful in preparing a library of potential binders corresponding to theselected parental template. The seven libraries discussed above wereprepared according to such techniques, and they were screened for KDR orVEGF/KDR complex binding polypeptides against an immobilized target, asexplained in the examples to follow.

In a typical screen, a phage library is contacted with and allowed tobind the target, or a particular subcomponent thereof. To facilitateseparation of binders and non-binders, it is convenient to immobilizethe target on a solid support. Phage bearing a target-binding moietyform a complex with the target on the solid support whereas non-bindingphage remain in solution and may be washed away with excess buffer.Bound phage are then liberated from the target by changing the buffer toan extreme pH (pH 2 or pH 10), changing the ionic strength of thebuffer, adding denaturants, or other known means. To isolate the bindingphage exhibiting the polypeptides of the present invention, a proteinelution was performed, i.e., some phage were eluted from target usingVEGF in solution (competitive elution); and also, very high affinitybinding phage that could not be competed off incubating with VEGFovernight were captured by using the phage still bound to substrate forinfection of E. coli cells.

The recovered phage may then be amplified through infection of bacterialcells and the screening process repeated with the new pool that is nowdepleted in non-binders and enriched in binders. The recovery of even afew binding phage is sufficient to carry the process to completion.After a few rounds of selection, the gene sequences encoding the bindingmoieties derived from selected phage clones in the binding pool aredetermined by conventional methods, described below, revealing thepeptide sequence that imparts binding affinity of the phage to thetarget. When the selection process works, the sequence diversity of thepopulation falls with each round of selection until desirable bindersremain. The sequences converge on a small number of related binders,typically 10-50 out of the more than 10 million original candidates fromeach library. An increase in the number of phage recovered at each roundof selection, and of course, the recovery of closely related sequencesare good indications that convergence of the library has occurred in ascreen. After a set of binding polypeptides is identified, the sequenceinformation may be used to design other secondary phage libraries,biased for members having additional desired properties.

Formation of the disulfide binding loop is advantageous because it leadsto increased affinity and specificity for such peptides. However, inserum, the disulfide bond might be opened by free cysteines or otherthiol-containing molecules. Thus, it may be useful to modify thecysteine residues to replace the disulfide cross-link with another lessreactive linkage. The —CH₂—S—S—CH₂-cross-link has a preferred geometryin which the dihedral bond between sulfurs is close to 90 degrees, butthe exact geometry is determined by the context of other side groups andthe binding state of the molecule. Preferred modifications of theclosing cross-link of the binding loop will preserve the overall bondlengths and angles as much as possible. Suitable such alternativecross-links include thioether linkages such as —CH₂—S—CH₂—CH₂—,—CH₂—CH₂—S—CH₂—, —CH₂—CH₂—S—CH₂—CH₂—; lactam linkages such as—CH₂—NH—CO—CH₂— and —CH₂—CO—NH—CH₂—; ether linkages such as—CH₂—CH₂—O—CH₂—CH₂—; alkylene bridges such as —(CH₂)_(n)— (where n=4, 5,or 6); the linkage —CH₂—NH—CO—NH—CH₂—, and similar groups known in theart.

Although polypeptides containing a stable disulfide-linked binding loopare most preferred, linear polypeptides derived from the foregoingsequences may be readily prepared, e.g., by substitution of one or bothcysteine residues, which may retain at least some of the KDR or VEGF/KDRbinding activity of the original polypeptide containing the disulfidelinkage. In making such substitutions for Cys, the amino acids Gly, Ser,and Ala are preferred, and it is also preferred to substitute both Cysresidues, so as not to leave a single Cys that may cause the polypeptideto dimerize or react with other free thiol groups in a solution. Allsuch linearized derivatives that retain KDR or VEGF/KDR bindingproperties are within the scope of this invention.

Direct synthesis of the polypeptides of the invention may beaccomplished using conventional techniques, including solid-phasepeptide synthesis, solution-phase synthesis, etc. Solid-phase synthesisis preferred. See Stewart et al., Solid-Phase Peptide Synthesis (W. H.Freeman Co., San Francisco, 1989); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963); Bodanszky and Bodanszky, The Practice of PeptideSynthesis (Springer-Verlag, New York, 1984), incorporated herein byreference.

Polypeptides according to the invention may also be preparedcommercially by companies providing peptide synthesis as a service(e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality ControlledBiochemicals, Inc., Hopkinton, Mass.).

Automated peptide synthesis machines, such as manufactured byPerkin-Elmer Applied Biosystems, also are available.

The polypeptide compound is preferably purified once it has beenisolated or synthesized by either chemical or recombinant techniques.For purification purposes, there are many standard methods that may beemployed, including reversed-phase high-pressure liquid chromatography(RP-HPLC) using an alkylated silica column such as C₄-, C₈- orC₁₈-silica. A gradient mobile phase of increasing organic content isgenerally used to achieve purification, for example, acetonitrile in anaqueous buffer, usually containing a small amount of trifluoroaceticacid. Ion-exchange chromatography can also be used to separate peptidesbased on their charge. The degree of purity of the polypeptide may bedetermined by various methods, including identification of a major largepeak on HPLC. A polypeptide that produces a single peak that is at least95% of the input material on an HPLC column is preferred. Even morepreferable is a polypeptide that produces a single peak that is at least97%, at least 98%, at least 99% or even 99.5% or more of the inputmaterial on an HPLC column.

In order to ensure that the peptide obtained using any of the techniquesdescribed above is the desired peptide for use in compositions of thepresent invention, analysis of the peptide composition may be carriedout. Such composition analysis may be conducted using high resolutionmass spectrometry to determine the molecular weight of the peptide.Alternatively, the amino acid content of the peptide can be confirmed byhydrolyzing the peptide in aqueous acid, and separating, identifying andquantifying the components of the mixture using HPLC, or an amino acidanalyzer. Protein sequenators, which sequentially degrade the peptideand identify the amino acids in order, may also be used to determine thesequence of the peptide.

KDR or VEGF/KDR complex binding polypeptides according to the presentinvention also may be produced using recombinant DNA techniques,utilizing nucleic acids (polynucleotides) encoding the polypeptidesaccording to this invention and then expressing them recombinantly,i.e., by manipulating host cells by introduction of exogenous nucleicacid molecules in known ways to cause such host cells to produce thedesired KDR or VEGF/KDR complex binding polypeptides. Such proceduresare within the capability of those skilled in the art (see Davis et al.,Basic Methods in Molecular Biology, (1986)), incorporated by reference.Recombinant production of short peptides such as those described hereinmay not be practical in comparison to direct synthesis, howeverrecombinant means of production may be very advantageous where a KDR orVEGF/KDR complex binding moiety of this invention is incorporated in ahybrid polypeptide or fusion protein.

In the practice of the present invention, a determination of theaffinity of the KDR or VEGF/KDR complex binding moiety for KDR orVEGF/KDR complex relative to another protein or target is a usefulmeasure, and is referred to as specificity for KDR or VEGF/KDR complex.Standard assays for quantitating binding and determining affinityinclude equilibrium dialysis, equilibrium binding, gel filtration, orthe monitoring of numerous spectroscopic changes (such as a change influorescence polarization) that may result from the interaction of thebinding moiety and its target. These techniques measure theconcentration of bound and free ligand as a function of ligand (orprotein) concentration. The concentration of bound polypeptide ([Bound])is related to the concentration of free polypeptide ([Free]) and theconcentration of binding sites for the polypeptide, i.e., on KDR orVEGF/KDR complex, (N), as described in the following equation:[Bound]=N×[Free]/((1/K_(a))+[Free]).

A solution of the data to this equation yields the association constant,K_(a), a quantitative measure of the binding affinity. The associationconstant, K_(a) is the reciprocal of the dissociation constant, K_(D).The K_(D) is more frequently reported in measurements of affinity.Preferred KDR or VEGF/KDR complex binding polypeptides have a K_(D) forKDR or VEGF/KDR complex in the range of 1 nanomolar (nM) to 100micromolar (μM), which includes K_(D) values of less than 10 nM, lessthan 20 nM, less than 40 nM, less than 60 nM, less than 80 nM, less than1 μM, less than 5 μM, less than 10 μM, less than 20 μM, less than 40 μM,less than 60 μM, and less than 80 μM.

Where KDR or VEGF/KDR complex binding moieties are employed as imagingagents, other aspects of binding specificity may become more important.Imaging agents operate in a dynamic system in that binding of theimaging agent to the target (KDR or VEGF/KDR complex, e.g., on activatedendothelium) may not be in a stable equilibrium state throughout theimaging procedure. For example, when the imaging agent is initiallyinjected, the concentration of imaging agent and of agent-target complexrapidly increases. Shortly after injection, however, the circulating(free) imaging agent starts to clear through the kidneys or liver, andthe plasma concentration of imaging agent begins to drop. This drop inthe concentration of free imaging agent in the plasma eventually causesthe agent-target complex to dissociate. The usefulness of an imagingagent depends on the difference in rate of agent-target dissociationrelative to the clearing rate of the agent. Ideally, the dissociationrate will be slow compared to the clearing rate, resulting in a longimaging time during which there is a high concentration of agent-targetcomplex and a low concentration of free imaging agent (backgroundsignal) in the plasma.

Quantitative measurement of dissociation rates may be easily performedusing several methods known in the art, such as fiber optic fluorimetry(see, e.g., Anderson & Miller, Clin. Chem., 34(7):1417-21 (1988)),surface plasmon resonance (see, Malmborg et al., J. Immunol. Methods,198(1):51-7 (1996) and Schuck, Current Opinion in Biotechnology,8:498-502 (1997)), resonant mirror, and grating coupled planarwaveguiding (see, e.g., Hutchinson, Molec. Biotechnology, 3:47-54(1995)). Automated biosensors are commercially available for measuringbinding kinetics: BIAcore surface plasmon resonance sensor (Biacore AB,Uppsala SE), IAsys resonant mirror sensor (Fisons Applied SensorTechnology, Cambridge GB), BIOS-1 grated coupled planar waveguidingsensor (Artificial Sensor Instruments, Zurich CH).

Methods of Screening Polypeptides Identified by Phage Display for theirAbility to Bind to Cells Expressing the Target:

In another aspect of the invention, methods of screening bindingpolypeptides identified by phage display for their ability to bind tocells expressing the target (and not to cells that do not express thetarget) are provided. These methods address a significant problemassociated with screening peptides identified by phage display:frequently the peptides so identified do not have sufficient affinityfor the target to be screened against target-expressing cells inconventional assays. However, ascertaining that a particularphage-identified peptide binds to cells that express the target (anddoes not bind to cells that do not) is a critical piece of informationin identifying binding peptides that are potential in vivo targetingmoieties. The method takes advantage of the increase in affinity andavidity associated with multivalent binding and permits screening ofpolypeptides with low affinities against target-expressing cells.

The method generally consists of preparation and screening of multimericconstructs including one or more binding polypeptides. For example,polypeptides identified by phage display as binding to a target arebiotinylated and complexed with avidin, streptavidin or neutravidin toform tetrameric constructs. These tetrameric constructs are thenincubated with cells that express the desired target and cells that donot, and binding of the tetrameric construct is detected. Binding may bedetected using any method of detection known in the art. For example, todetect binding the avidin, streptavidin, or neutravidin may beconjugated to a detectable marker (e.g., a radioactive label, afluorescent label, or an enzymatic label that undergoes a color change,such as HRP (horse radish peroxidase), TMB (tetramethyl benzidine) oralkaline phosphatase).

The biotinylated peptides are preferably complexed with neutravidin-HRP.Neutravidin exhibits lower non-specific binding to molecules than theother alternatives due to the absence of lectin binding carbohydratemoieties and cell adhesion receptor-binding RYD domain in neutravidin.See, Hiller et al., Biochem. J., 248:167-171 (1987); Alon et al.,Biochem. Biophys. Res. Commun., 170:1236-41 (1990).

The tetrameric constructs can be screened against cells that naturallyexpress the target or cells that have been engineered via recombinantDNA technologies to express the target (e.g., transfectants,transformants, etc.). If cells that have been transfected to express thetarget are used, mock-transfected cells (i.e., cells transfected withoutthe genetic material encoding the target) may be used as a control.

The tetrameric complexes may optionally be screened in the presence ofserum. Thus, the assay may also be used to rapidly evaluate the effectof serum on the binding of peptides to the target.

The methods disclosed herein are particularly useful in preparing andevaluating combinations of distinct binding polypeptides for use indimeric or multimeric targeting contructs that contain two or morebinding polypeptides. Use of biotin/avidin complexes allows forrelatively easy preparation of tetrameric constructs containing one tofour different binding peptides. Furthermore, it has now been found thataffinity and avidity of a targeting construct may be increased byinclusion of two or more targeting moieties that bind to differentepitopes on the same target. The screening methods described herein areuseful in identifying combinations of binding polypeptides that may haveincreased affinity when included in such multimeric constructs.

In a preferred embodiment, the screening methods described herein may beused to screen KDR and VEGF/KDR complex binding polypeptides identifiedby phage display, such as those described herein. As described in moredetail in Example 5 infra, these methods may be used to assess thespecific binding of KDR binding polypeptides to cells that express KDRor have been engineered to express KDR. Tetrameric complexes ofbiotinylated KDR binding polypeptides of the invention andneutravidin-HRP may be prepared and screened against cells transfectedto express KDR as well as mock transfected cells (without any KDR).

As shown in Example 5, the assay can be used to identify KDR bindingpolypeptides that bind specifically to KDR-expressing cells (and do notbind to cells that do not express KDR) even when the monodentate K_(D)of the polypeptide is on the order of 200 nM-300 nM. The assay may beused to screen homotetrameric constructs containing four copies of asingle KDR binding polypeptide of the invention as well asheterotetrameric constructs (e.g., constructs containing two or moredifferent KDR binding polypeptides). The methods described herein areparticularly useful for assessing combinations of KDR bindingpolypeptides for use in multimeric constructs, particularly constructscontaining two or more KDR binding polypeptides that bind to differentepitopes of KDR.

The assay may also be used to assess the effect of serum on the KDRbinding polypeptides. Indeed, using the screening methods disclosedherein, KDR binding polypeptides, such as SEQ ID NOS: 264, 294, and 356,were identified whose binding is not significantly affected by serum.

Modification or Optimization of KDR and VEGF/KDR Complex BindingPolypeptides

As discussed, modification or optimization of KDR and VEGF/KDR complexbinding polypeptides is within the scope of the invention and themodified or optimized polypeptides are included within the definition of“KDR and VEGF/KDR complex binding polypeptides”. Specifically, apolypeptide sequence identified by phage display can be modified tooptimize its potency, pharmacokinetic behavior, stability and/or otherbiological, physical and chemical properties.

Substitution of Amino Acid Residues

For example, one can make the following isosteric and/or conservativeamino acid changes in the parent polypeptide sequence with theexpectation that the resulting polypeptides would have a similar orimproved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: Includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from C₁₋₁₀ carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: Includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy (from C1-C4)-substituted forms of theprevious listed aromatic amino acids, illustrative examples of whichare: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine,2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine,5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or4′-amino-, 2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′, -3′,-or 4′-methyl-2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine

Substitution of amino acids containing basic functions: Includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from C1-C10 branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma′-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known activated derivatives such as acidchlorides, active esters, active azolides and related derivatives) andlysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: Including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: Including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.

Substitution of hydroxyl containing amino acids: Including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine. It is also understoodthat the amino acids within each of the categories listed above may besubstituted for another of the same group.

Substitution of Amide Bonds

Another type of modification within the scope of the patent is tosubstitute the amide bonds within the backbone of the polypeptide. Forexample, to reduce or eliminate undesired proteolysis, or otherdegradation pathways that diminish serum stability, resulting in reducedor abolished bioactivity, or to restrict or increase conformationalflexibility, it is common to substitute amide bonds within the backboneof the peptides with functionality that mimics the existing conformationor alters the conformation in the manner desired. Such modifications mayproduce increased binding affinity or improved pharmacokinetic behavior.It is understood that those knowledgeable in the art of peptidesynthesis can make the following amide bond-changes for any amide bondconnecting two amino acids with the expectation that the resultingpeptides could have the same or improved activity: insertion ofalpha-N-methylamides or peptide amide backbone thioamides, removal ofthe carbonyl to produce the cognate secondary amines, replacement of oneamino acid with an aza-aminoacid to produce semicarbazone derivatives,and use of E-olefins and substituted E-olefins as amide bond surrogates.

Introduction of D-Amino Acids

Another approach within the scope of the patent is the introduction ofD-alanine, or another D-amino acid, distal or proximal to the labilepeptide bond. In this case it is also understood to those skilled in theart that such D-amino acid substitutions can, and at times, must bemade, with D-amino acids whose side chains are not conservativereplacements for those of the L-amino acid being replaced. This isbecause of the difference in chirality and hence side-chain orientation,which may result in the accessing of a previously unexplored region ofthe binding site of the target that has moieties of different charge,hydrophobicity, steric requirements etc. than that serviced by the sidechain of the replaced L-amino acid.

Modifications to Improve Pharmacokinetic or Pharmacodynamic Properties

It is also understood that use of the KDR or VEGF/KDR complex bindingpolypeptide in a particular application may necessitate modifications ofthe peptide or formulations of the peptide to improve pharmacokineticand pharmacodynamic behavior. It is expected that the properties of thepeptide may be changed by attachment of moieties anticipated to bringabout the desired physical or chemical properties. Such moieties may beappended to the peptide using acids or amines, via amide bonds or ureabonds, respectively, to the N- or C-terminus of the peptide, or to thependant amino group of a suitably located lysine or lysine derivative,2,3-diaminopropionic acid, ornithine, or other amino acid in the peptidethat possesses a pendant amine group or a pendant alkoxyamine orhydrazine group. The moieties introduced may be groups that arehydrophilic, basic, or nonpolar alkyl or aromatic groups depending onthe peptide of interest and the extant requirements for modification ofits properties.

Glycosylation of Amino Acid Residues

Yet another modification within the scope of the invention is to employglycosylated amino acid residues (e.g., serine, threonine or asparagineresidues), singly or in combination in the either the binding moiety (ormoieties) or the linker moiety or both. Glycosylation, which may becarried out using standard conditions, can be used to enhancesolubility, alter pharmacokinetics and pharmacodynamics or to enhancebinding via a specific or non-specific interaction involving theglycosidic moiety. In another approach glycosylated amino acids such asO-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl) serine orthe analogous threonine derivative (either the D- or L-amino acids) canbe incorporated into the peptide during manual or automated solid phasepeptide synthesis, or in manual or automated solution phase peptidesynthesis. Similarly D- orL-N^(γ)-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-asparaginecan be employed. The use of amino acids glycosylated on a pendantoxygen, nitrogen or sulfur function by the agency of suitablyfunctionalized and activated carbohydrate moieties that can be employedin glycosylation is anticipated. Such carbohydrate functions could bemonosaccharides, disaccharides or even larger assemblies ofoligosaccharides (Kihlberg, January (2000) Glycopeptide synthesis. In:Fmoc Solid Phase Peptide Synthesis—A Practical Approach (Chan, W. C. andWhite, P. D. Eds) Oxford University Press, New York, N.Y. Chap. 8, pp195-213).

Also anticipated is the appendage of carbohydrate functions to aminoacids by means other than glycosylation via activation of a leavinggroup at the anomeric carbon. Linkage of the amino acid to the glycosideis not limited to the formation of a bond to the anomeric carbon of thecarbohydrate function. Instead, linkage of the carbohydrate moiety tothe amino acid could be through any suitable, sufficiently reactiveoxygen atom, nitrogen atom, carbon atom or other pendant atom of thecarbohydrate function via methods employed for formation ofC-heteroatom, C—C or heteroatom-heteroatom (examples are S—S, O—N, N—N,P—O, P—N) bonds known in the art.

Formation of Salts

It is also within the scope of the invention to form different saltsthat may increase the water solubility or the ease of formulation ofthese peptides. These may include, but are not restricted to,N-methylglucamine (meglumine), acetate, oxalates, ascorbates, etc.

Structural Modifications that Retain Structural Features

Yet another modification within the scope of the invention is truncationof cyclic polypeptides. The cyclic nature of many polypeptides of theinvention limits the conformational space available to the peptidesequence, particularly within the cycle. Therefore truncation of thepeptide by one or more residues distal or even proximal to the cycle, ateither the N-terminal or C-terminal region may provide truncatedpeptides with similar or improved biological activity. A unique sequenceof amino acids, even as small as three amino acids, which is responsiblefor the binding activity, may be identified, as noted for RGD peptides(see, e.g., Plow et al., Blood, 70(1): 110-5 (1987); Oldberg et al.,Journal of Biological Chemistry, 263(36):19433-19436 (1988); Taub etal., Journal of Biological Chemistry, 264(1):259-65 (1989); Andrieux etal., Journal of Biological Chemistry, 264(16):9258-65 (1989); and U.S.Pat. No. 5,773,412 and U.S. Pat. No. 5,759,996, each of which isincorporated herein by reference).

It has also been shown in the literature that large peptide cycles canbe substantially shortened, eliminating extraneous amino acids, butsubstantially including the critical binding residues. See, U.S. Pat.No. 5,556,939, incorporated by reference herein.

The shortened cyclic peptides can be formed using disulfide bonds oramide bonds of suitably located carboxylic acid groups and amino groups.

Furthermore, D-amino acids can be added to the peptide sequence tostabilize turn features (especially in the case of glycine). In anotherapproach alpha, beta, gamma or delta dipeptide or turn mimics (such asα, β, γ, or δ turn mimics), some of which are shown in schematics 1, 2and 3 as shown in FIG. 26, can be employed to mimic structural motifsand turn features in a peptide and simultaneously provide stability fromproteolysis and enhance other properties such as, for example,conformational stability and solubility (structure 1: Hart et al., J.Org. Chem., 64, 2998-2999 (1999); structure 2: Hanessian et al.,“Synthesis of a Versatile Peptidomimetic Scaffold” in Methods inMolecular Medicine, Vol. 23: Peptidomimetics Protocols, W. M.Kazmierski, Ed. (Humana Press Inc., Totowa, N.J., 1999), Chapter 10, pp.161-174; structure 3: WO 01/16135).

Substitution of Disulfide Mimetics

Also within the scope of the invention is the substitution of disulfidemimetics for disulfide bonds within the KDR or VEGF/KDR complex bindingpeptides of the invention.

When disulfide-containing peptides are employed in generating^(99m)Tc-based radiopharmaceuticals, a significant problem is thepresence of the disulfide bond. The integrity of the disulfide bond isdifficult to maintain during procedures designed to incorporate ^(99m)Tcvia routes that are reliant upon the reduction of pertechnetate ion andsubsequent incorporation of the reduced Tc species into substancesbearing Tc-specific chelating groups. This is because the disulfide bondis rather easily reduced by the reducing agents commonly used in kitsdevised for one-step preparation of radiopharmaceuticals. Therefore, theease with which the disulfide bond can be reduced during Tc chelationmay require substitution with mimetics of the disulfide bonds.Accordingly, another modification within the scope of the invention isto substitute the disulfide moiety with mimetics utilizing the methodsdisclosed herein or known to those skilled in the art, while retainingthe activity and other desired properties of the KDR-bindingpolypeptides of the invention:

1) Oxime Linker

The oxime moiety has been employed as a linker by investigators in anumber of contexts. Of the most interest is the work by Mutter et al.(Wahl and Mutter, Tetrahedron Lett., 37:6861-6864 (1996)). The aminoacids 4, containing an aminoalcohol function, and 5, containing analkoxyamino function, are incorporated into the peptide chain, notnecessarily at the end of the peptide chain (FIG. 27). After formationof the peptide the sidechain protecting groups are removed. The aldehydegroup is unmasked and an oxime linkage is formed.

2) Lanthionine Linker

Lanthionines are cyclic sulfides, wherein the disulfide linkage (S—S) isreplaced by a carbon-sulfur (C—S) linkage. Thus, the lability toreduction is far lower. Lanthionines have been prepared by a number ofmethods since 1971.

Preparation of Lanthionines Using Bromoacetylated Peptides

Lanthionines are readily prepared using known methods. See, for example,Robey et al., Anal. Biochem., 177:373-377 (1989); Inman et al.,Bioconjugate Chem., 2:458-463 (1991); Ploinsky et al., Med. Chem.,35:4185-4194 (1992); Mayer et al., “Peptides, Frontiers of PeptideScience”, in Proceedings of the 15^(th) American Peptide Symposium, Tam& Kaumaya (Eds.), Jun. 14-19, 1995, Nashville, Tenn. (Klumer AcademicPub., Boston), pp. 291-292; Wakao et al., Jpn. Kokai Tokyo Koho, JP07300452 A2 (1995). Preparation of peptides using Boc automated peptidesynthesis followed by coupling the peptide terminus with bromoaceticacid gives bromoacetylated peptides in good yield. Cleavage anddeprotection of the peptides is accomplished using HF/anisole. If thepeptide contains a cysteine group its reactivity can be controlled withlow pH. If the pH of the medium is raised to 6-7 then eitherpolymerization or cyclization of the peptide takes place. Polymerizationis favored at high (100 mg/mL) concentration whereas cyclization isfavored at lower concentrations (1 mg/mL), e.g., 6 cyclizes to 7 (Scheme1; FIG. 28).

Inman et al. demonstrated the use ofN^(α)-(Boc)-N^(ε)-[N-(bromoacetyl)-β-alanyl]-L-lysine as a carrier ofthe bromoacetyl group that could be employed in Boc peptide synthesisthus allowing placement of a bromoacetyl bearing moiety anywhere in asequence. In preliminary experiments they found that peptides with 4-6amino acids separating the bromoacetyl-lysine derivative from a cysteinetend to cyclize, indicating the potential utility of this strategy.

Preparation of Lanthionines Via Cysteine Thiol Addition to Acrylamides

Several variants of this strategy may be implemented. Resin-bound serinecan be employed to prepare the lanthionine ring on resin either using abromination-dehydrobromination-thiol addition sequence or by dehydrationwith disuccinimidyl carbonate followed by thiol addition (Ploinsky etal., M. J. Med. Chem., 35:4185-4194 (1992); Mayer et al., “Peptides,Frontiers of Peptide Science”, in Proceedings of the 15^(th) AmericanPeptide Symposium, Tam & Kaumaya (Eds.), Jun. 14-19, 1995, Nashville,Tenn. (Klumer Academic Pub., Boston), pp. 291-292). Conjugate additionof thiols to acrylamides has also been amply demonstrated and areference to the addition of 2-mercaptoethanol to acrylamide is provided(Wakao et al., Jpn. Kokai Tokyo Koho, JP 07300452 A2 (1995)).

3) Diaryl Ether or Diarylamine Linkage: Diaryl Ether Linkage fromIntramolecular Cyclization of Aryl Boronic Acids and Tyrosine

Recently the reaction of arylboronic acids with phenols, amines andheterocyclic amines in the presence of cupric acetate, in air, atambient temperature, in dichloromethane using either pyridine ortriethylamine as a base to provide unsymmetrical diaryl ethers and therelated amines in good yields (as high as 98%) has been reported. See,Evans et al., Tetrahedron Lett., 39:2937-2940 (1998); Chan et al.,Tetrahedron Lett., 39:2933-2936 (1998); Lam et al., Tetrahedron Lett.,39:2941-2944 (1998). In the case of N-protected tyrosine derivatives asthe phenol component the yields were also as high as 98%. Thisdemonstrates that amino acid amides (peptides) are expected to be stableto the transformation and that yields are high. Precedent for anintramolecular reaction exists in view of the facile intramolecularcyclizations of peptides to lactams, intramolecular biaryl etherformation based on the S_(N)Ar reaction and the generality ofintramolecular cyclization reactions under high dilution conditions oron resin, wherein the pseudo-dilution effect mimics high dilutionconditions.

4) Formation of Cyclic Peptides with a Thiazolidine Linkage viaIntramolecular Reaction of Peptide Aldehydes with Cysteine Moieties

Another approach that may be employed involves intramolecularcyclization of suitably located vicinal amino mercaptan functions(usually derived from placement of a cysteine at a terminus of thelinear sequence or tethered to the sequence via a side-chain nitrogen ofa lysine, for example) and aldehyde functions to provide thiazolidinesthat result in the formation of a bicyclic peptide, one ring of which isthat formed by the residues in the main chain, and the second ring beingthe thiazolidine ring. Scheme 2 (FIG. 29) provides an example. Therequired aldehyde function can be generated by sodium metaperiodatecleavage of a suitably located vicinal aminoalcohol function, which canbe present as an unprotected serine tethered to the chain by appendageto a side chain amino group of a lysine moiety. In some cases therequired aldehyde function is generated by unmasking of a protectedaldehyde derivative at the C-terminus or the N-terminus of the chain. Anexample of this strategy is found in: Botti et al., J. Am. Chem. Soc.,118:10018-10034 (1996).

5) Lactams Based on Intramolecular Cyclization of Pendant Amino Groupswith Carboxyl Groups on Resin.

Macrocyclic peptides have been prepared by lactam formation by eitherhead to tail or by pendant group cyclization. The basic strategy is toprepare a fully protected peptide wherein it is possible to removeselectively an amine protecting group and a carboxy protecting group.Orthogonal protecting schemes have been developed. Of those that havebeen developed the allyl, trityl and Dde methods have been employedmost. See, Mellor et al., “Synthesis of Modified Peptides”, in FmocSolid Phase Synthesis: A Practical Approach, White and Chan (eds)(Oxford University Press, New York, 2000), Chapt. 6, pp. 169-178. TheDde approach is of interest because it utilizes similar protectinggroups for both the carboxylic acid function (Dmab ester) and the aminogroup (Dde group). Both are removed with 2-10% hydrazine in DMF atambient temperature. Alternatively, the Dde can be used for the aminogroup and the allyl group can be used for the carboxyl.

A lactam function, available by intramolecular coupling via standardpeptide coupling reagents (such as HATU, PyBOP etc), could act as asurrogate for the disulfide bond. The Dde/Dmab approach is shown in FIG.30.

Thus, a linear sequence containing, for example, the Dde-protectedlysine and Dmab ester can be prepared on a Tentagel-based Rink amideresin at low load (˜0.1-0.2 mmol/g). Deprotection of both functions withhydrazine is then followed by on-resin cyclization to give the desiredproducts.

In the allyl approach, shown in FIG. 31, the pendant carboxyl that is toundergo cyclization is protected as an allyl ester and the pendant aminogroup is protected as an alloc group. On resin, both are selectivelyunmasked by treatment with palladium tris-triphenylphosphine in thepresence of N-methylmorpholine and acetic acid in DMF. Residualpalladium salts are removed using sodium diethyldithiocarbamate in thepresence of DIEA in DMF, followed by subsequent washings with DMF. Thelactam ring is then formed employing HATU/HOAt in the presence ofN-methylmorpholine. Other coupling agents can be employed as describedabove. The processing of the peptide is then carried out as describedabove to provide the desired peptide lactam.

Subsequently cleavage from resin and purification can also be carriedout. For functionalization of the N-terminus of the peptide, it isunderstood that amino acids, such astrans-4-(iV-Dde)methylaminocyclohexane carboxylic acid,trans-4-(iV-Dde)methylaminobenzoic acid, or their alloc congeners can beemployed. Yet another approach is to employ the safety catch method tointramolecular lactam formation during cleavage from the resin.

Thus, a linear sequence containing, for example, the Dde-protectedlysine and Dmab ester may be prepared on a Tentagel-based Rink amideresin at low load (˜0.1-0.2 mmol/g). Deprotection of both functions withhydrazine is then followed by on-resin cyclization to give the desiredproducts. Subsequently cleavage from resin and purification may also becarried out. For functionalization of the N-terminus of the peptide itis understood that diamino acids such astrans-4-(iv-Dde)methylaminocyclohexane carboxylic acid ortrans-4-(iv-Dde)methylamino benzoic acid would be required. Analternative scenario is to employ the safety catch method tointramolecular lactam formation during cleavage from the resin.

6) Cyclic Peptides Based on Olefin Metathesis

The Grubbs reaction (FIG. 32) involves the metathesis/cyclization ofolefin bonds and is illustrated as shown below. See, Schuster et al.,Angewandte. Chem. Int. Edn Engl., 36:2036-2056 (1997); Miller et al., J.Am. Chem. Soc., 118:9606-9614 (1996).

It is readily seen (FIG. 32) that if the starting material is a diolefin(16) that the resulting product will be cyclic compound 17. The reactionhas in fact been applied to creation of cycles fromolefin-functionalized peptides. See, e.g., Pernerstorfer et al., Chem.Commun., 20:1949-50 (1997); see, also, Covalent capture andstabilization of cylindrical β-sheet peptide assemblies, Clark et al.,Chem. Eur. J., 5(2):782-792 (1999); Highly efficient synthesis ofcovalently cross-linked peptide helices by ring-closing metathesis,Blackwell et al., Angew. Chem., Int. Ed., 37(23):3281-3284 (1998);Synthesis of novel cyclic protease inhibitors using Grubbs olefinmetathesis, Ripka et al., Med. Chem. Lett., 8(4):357-360 (1998);Application of Ring-Closing Metathesis to the Synthesis of RigidifiedAmino Acids and Peptides, Miller et al., J. Am. Chem. Soc.,118(40):9606-9614 (1996); Supramolecular Design by Covalent Capture,Design of a Peptide Cylinder via Hydrogen-Bond-Promoted IntermolecularOlefin Metathesis, Clark et al., J. Am. Chem. Soc., 117(49):12364-12365(1995); Synthesis of Conformationally Restricted Amino Acids andPeptides Employing Olefin Metathesis, Miller et al., J. Am. Chem. Soc.,117(21):5855-5856 (1995). One can prepare either C-allylated amino acidsor possibly N-allylated amino acids and employ them in this reaction inorder to prepare carba-bridged cyclic peptides as surrogates fordisulfide bond containing peptides.

One may also prepare novel compounds with olefinic groups.Functionalization of the tyrosine hydroxyl with an olefin-containingtether is one option. The lysine ε-amino group is another option withappendage of the olefin-containing unit as part of an acylating moiety,for example. If instead the lysine side chain amino group is alkylatedwith an olefin containing tether, it can still function as a point ofattachment for a reporter as well. The use of 5-pentenoic acid as anacylating agent for the lysine, ornithine, or diaminopropionic sidechain amino groups is another possibility. The length of theolefin-containing tether can also be varied in order to explorestructure activity relationships.

Manipulation of Peptide Sequences

Other modifications within the scope of the invention include commonmanipulations of peptide sequences, which can be expected to yieldpeptides with similar or improved biological properties. These includeamino acid translocations (swapping amino acids in the sequence), use ofretroinverso peptides in place of the original sequence or a modifiedoriginal sequence, peptoids and retro-inverso peptoid sequences.Structures wherein specific residues are peptoid instead of peptidic,which result in hybrid molecules, neither completely peptidic norcompletely peptoid, are anticipated as well.

Linkers

Additional modifications within the scope of the invention includeintroduction of linkers or spacers between the targeting sequence of theKDR or VEGF/KDR complex binding peptide and the detectable label ortherapeutic agent. Use of such linkers/spacers may improve the relevantproperties of the binding peptide (e.g., increase serum stability,etc.). These linkers may include, but are not restricted to, substitutedor unsubstituted alkyl chains, polyethylene glycol derivatives, aminoacid spacers, sugars, or aliphatic or aromatic spacers common in theart. Furthermore, linkers that are combinations of the moietiesdescribed above, can also be employed to confer special advantage to theproperties of the peptide. Lipid molecules with linkers may be attachedto allow formulation of ultrasound bubbles, liposomes or otheraggregation based constructs. Such constructs could be employed asagents for targeting and delivery of a diagnostic reporter, atherapeutic agent (e.g., a chemical “warhead” for therapy) or acombination of these.

Multimeric Constructs of KDR and VEGF/KDR Complex Binding Polypeptides

Constructs employing dimers, multimers or polymers of one or more VEGFor VEGF/KDR complex binding polypeptides of the invention are alsocontemplated. Indeed, there is ample literature evidence that thebinding of low potency peptides or small molecules can be substantiallyincreased by the formation of dimers and multimers. Thus, dimeric andmultimeric constructs (both homogeneous and heterogeneous) are withinthe scope of the instant invention. Indeed, as discussed in more detailin the Examples, it is within the scope of the present invention toinclude multiple KDR or VEGF/KDR complex binding polypeptide sequencesin a dimeric or multimeric construct. Moreover, as shown in Example 4infra, these constructs can exhibit improved binding compared to amonomeric construct. The polypeptide sequences in the dimeric constructsmay be attached at their N- or C-terminus or the N-epsilon nitrogen of asuitably placed lysine moiety (or another function bearing a selectivelyderivatizable group such as a pendant oxyamino or other nucleophilicgroup), or may be joined together via one or more linkers employing theappropriate attachment chemistry. This coupling chemistry may includeamide, urea, thiourea, oxime, or aminoacetylamide (from chloro- orbromoacetamide derivatives, but is not so limited. For example, any ofthe following methods may be utilized to prepare dimeric or multimericconstructs of KDR or VEGF/KDR complex binding polypeptides of theinvention. Modified polypeptides and peptide-derived molecules areshown, for example, in FIGS. 79A-79G.

Method A

Fully protected KDR-binding peptides can be built up on Ellman-typesafety catch resin using automated or manual Fmoc peptide synthesisprotocols. Backes et al., J. Am. Chem. Soc., 118(12):3055-56 (1996).Separately, using standard methods known in the art of peptidesynthesis, a di-lysine derivative can be constructed on 2-chlorotritylresin. See, for example, Fields et al, “Principles and Practice of SolidPhase Synthesis” in Synthetic Peptides, A Users Guide, Grant, Ed. (W.H.Freeman Co., New York, 1992), Chapt. 3, pp. 77-183; Barlos et al.,“Convergent Peptide Synthesis” in Fmoc Solid Phase Peptide Synthesis,Chan, W. C. and White, P. D., Eds. (Oxford University Press, New York,2000), Chapt. 9, pp. 215-228. Liberation of this from the 2-chlorotritylresin without removal of the side-chain protecting groups, activation ofthe carboxyl group and coupling to any amine-functionalized labelinggroup provides a di-lysine derivative whose protected pendant nitrogenatoms may be unmasked to give two free amino groups. The prior-mentionedsafety-catch resin is activated and the desired N-deprotected labelinggroup-functionalized di-lysine derivative is added to the activatedsafety-catch resin. The pendant amino groups are acylated by thecarboxy-terminus of the safety-catch resin-bound peptide, which is nowdetached from the resin and an integral part of the di-lysine structure.An excess of the safety-catch resin-bound peptide can be employed toinsure complete reaction of the amino groups of the di-lysine construct.Optimization of the ratio of the reacting partners in this schemeoptimizes the yield. The protecting groups on the KDR-binding peptidesare removed employing trifluoroacetic acid based cleavage protocols.

The synthesis of dimeric and multimeric constructs wherein two or moreKDR-binding peptides are present in one construct is easilyaccomplished. Orthogonal protection schemes (such as an allyloxycarbonylgroup on one nitrogen and an Fmoc group on the other, or employing theFmoc group in conjunction with the iV-Dde protecting group on the other,for example) can be employed to distinguish the pendant nitrogen atomsof the di-lysine derivatives described above. Unmasking of one of theamino groups, followed by reaction of the resulting product with anactivated safety-catch resin-bound KDR-binding peptide as describedabove, provides a di-lysine construct having a single KDR-bindingpeptide attached. Removal of the second protecting group unmasks theremaining nitrogen. See, also, Mellor et al., “Synthesis of ModifiedPeptides” in Fmoc Solid Phase Peptide Synthesis, Chan, W. C. and White,P. D., Eds. (Oxford University Press, New York, 2000), Chapt. 6, pp.169-176. The resulting product may be reacted with a second safety-catchresin bearing another KDR-binding peptide to provide a fully-protectedhomodimeric construct, which after removal of protecting groups withtrifluoroacetic acid, provides the desired material.

Method B

A KDR-binding peptide is assembled on a Rink-amide resin by automated ormanual peptide coupling methods, usually employing Fmoc peptidesynthesis protocols. The peptide may possess a C-terminus or N-terminusfunctionalized with a linker or a linker-labeling group construct thatmay possess an additional nucleophilic group such as the ε-amino groupof a lysine moiety, for example. Cleavage of the protecting groups isaccomplished employing trifluoroacetic acid with appropriate modifiersdepending on the nature of the peptide. The fully deprotected peptide isthen reacted with a large excess of a bifunctional electrophile such asthe commercially available glutaric acid bis-N-hydroxysuccinimide ester(Tyger Scientific, Inc.). The resulting monoamidated,mono-N-hydroxysuccinimidyl ester of glutaric acid is then treated withan additional equivalent of the same peptide, or an equivalent of adifferent KDR-binding peptide. Purification of the resulting material byHPLC affords the desired homodimeric construct bearing a suitablelabeling group.

Method C

A modular scheme can be employed to prepare dimeric or higher multimericconstructs bearing suitable labeling groups as defined above. In asimple illustration, fmoc-lysine(iV-Dde) Rink amide resin is treatedwith piperidine to remove the fmoc moiety. Then a labeling function,such as biotin, 5-carboxyfluorescein orN,N-Dimethyl-Gly-Ser(O-t-Bu)-Cys(Acm)-Gly-OH is coupled to the nitrogenatom. The resin is next treated with hydrazine to remove the iV-Ddegroup. After thorough washing, the resin is treated with cyanuricchloride and a hindered base such as diisopropylethylamine in a suitablesolvent such as DMF, NMP or dichloromethane to provide amonofunctionalized dichlorotriazine bound to the resin. Subsequentsuccessive displacement of the remaining chlorine atoms by twoequivalents of a KDR-binding peptide provides a resin-bound homo-dimericlabeling group-functionalized construct. Falorni et al., TetrahedronLett., 39(41):7607-7610 (1998); Johnson et al., Tetrahedron Lett.,54(16):4097-4106 (1998); Stankova et al., Mol. Diversity, 2(1/2):75-80(1996). The incoming peptides may be protected or unprotected as thesituation warrants. Cleavage of protecting groups is accomplishedemploying trifluoroacetic acid-based deprotection reagents as describedabove, and the desired materials are purified by high performance liquidchromatography.

It is understood that in each of these methods lysine derivatives may beserially employed to increase the multiplicity of the multimers. The useof related, more rigid molecules bearing the requisite number of masked,or orthogonally protected nitrogen atoms to act as scaffolds to vary thedistance between the KDR-binding peptides, to increase the rigidity ofthe construct (by constraining the motion and relative positions of theKDR-binding peptides relative to each other and the reporter) isentirely within the scope of methods A-C and all other methods describedherein. The references cited above are incorporated by reference hereinin their entirety.

Uses for KDR or VEGF/KDR Complex Binding Polypeptides:

The KDR or VEGF/KDR complex binding moieties according to this inventionwill be extremely useful for detection and/or imaging of KDR or VEGF/KDRcomplex in vitro or in vivo, and particularly for detection and/orimaging of sites of angiogenesis, in which VEGF and KDR are intimatelyinvolved, as explained above. Any suitable method of assaying or imagingKDR or VEGF/KDR complex may be employed. The KDR and VEGF/KDR complexbinding moieties of the invention also have utility in the treatment ofa variety of disease states, including those associated withangiogenesis or those associated with a number of pathogens. The KDR andVEGF/KDR complex binding moieties of the invention may themselves beused as therapeutics or may be used to localize one or more therapeuticagents (e.g., a chemotherapeutic, a radiotherapeutic, genetic material,etc.) to KDR expressing cells, including sites of angiogenesis.

In Vitro:

For detection of KDR or VEGF/KDR complex in solution, a bindingpolypeptide according to the invention can be detectably labeled, e.g.,fluorescently labeled, enzymatically labeled, or labeled with aradioactive or paramagnetic metal, then contacted with the solution, andthereafter formation of a complex between the binding polypeptide andthe KDR or VEGF/KDR complex target can be detected. As an example, afluorescently labeled KDR or VEGF/KDR complex binding peptide may beused for in vitro KDR or VEGF/KDR complex detection assays, wherein thepeptide is added to a solution to be tested for KDR or VEGF/KDR complexunder conditions allowing binding to occur. The complex between thefluorescently labeled KDR or VEGF/KDR complex binding peptide and KDR orVEGF/KDR complex target can be detected and quantified by measuring theincreased fluorescence polarization arising from the KDR or VEGF/KDRcomplex-bound peptide relative to that of the free peptide.

Alternatively, a sandwich-type “ELISA” assay may be used, wherein a KDRor VEGF/KDR complex binding polypeptide is immobilized on a solidsupport such as a plastic tube or well, then the solution suspected ofcontaining KDR or VEGF/KDR complex target is contacted with theimmobilized binding moiety, non-binding materials are washed away, andcomplexed polypeptide is detected using a suitable detection reagent,such as a monoclonal antibody recognizing KDR or VEGF/KDR complex. Themonoclonal antibody is detectable by conventional means known in theart, including being detectably labeled, e.g., radiolabeled, conjugatedwith an enzyme such as horseradish peroxidase and the like, orfluorescently labeled, etc.

For detection or purification of soluble KDR or VEGF/KDR complex in orfrom a solution, binding polypeptides of the invention can beimmobilized on a solid substrate such as a chromatographic support orother matrix material, then the immobilized binder can be loaded orcontacted with the solution under conditions suitable for formation of abinding polypeptide: KDR complex or binding polypeptide:VEGF/KDRcomplex. The non-binding portion of the solution can be removed and thecomplex may be detected, e.g., using an anti-KDR or anti-VEGF/KDRcomplex antibody, or an anti-binding polypeptide antibody, or the KDR orVEGF/KDR complex target may be released from the binding moiety atappropriate elution conditions.

The biology of angiogenesis and the roles of VEGF and KDR in initiatingand maintaining it have been investigated by many researchers andcontinues to be an active field for research and development. Infurtherance of such research and development, a method of purifying bulkamounts of KDR or VEGF/KDR complex in pure form is desirable, and thebinding polypeptides according to this invention are especially usefulfor that purpose, using the general purification methodology describedabove.

In Vivo:

Diagnostic Imaging

A particularly preferred use for the polypeptides according to thepresent invention is for creating visually readable images of KDRexpressing tissue, such as, for example, neoplastic tumors, whichrequire angiogenesis for survival and metastasis, or other sites ofangiogenic activity. The KDR and VEGF/KDR complex binding polypeptidesdisclosed herein may be converted to imaging reagents by conjugating thepolypeptides with a label appropriate for diagnostic detection,optionally via a linker. Preferably, a peptide exhibiting much greaterspecificity for KDR or VEGF/KDR complex than for other serum proteins isconjugated or linked to a label appropriate for the detectionmethodology to be employed. For example, the KDR or VEGF/KDR complexbinding polypeptide may be conjugated with or without a linker to aparamagnetic chelate suitable for magnetic resonance imaging (MRI), witha radiolabel suitable for x-ray, PET or scintigrapic imaging (includinga chelator for a radioactive metal), with an ultrasound contrast agent(e.g., a stabilized microbubble, a ultrasound contrast agent, amicrosphere or what has been referred to as a gas filled “liposome”)suitable for ultrasound detection, or with an optical imaging dye.

Suitable linkers can be substituted or unsubstituted alkyl chains, aminoacid chains (e.g., polyglycine), polyethylene glycols, polyamides, andother simple polymeric linkers known in the art.

In general, the technique of using a detectably labeled KDR or VEGF/KDRcomplex binding moiety is based on the premise that the label generatesa signal that is detectable outside the patient's body. For example,when the detectably labeled KDR or VEGF/KDR complex binding moiety isadministered to the patient in which it is desirable to detect, e.g.,angiogenesis, the high affinity of the KDR or VEGF/KDR complex bindingmoiety for KDR or VEGF/KDR complex causes the binding moiety to bind tothe site of angiogenesis and accumulate label at the site ofangiogenesis. Sufficient time is allowed for the labeled binding moietyto localize at the site of angiogenesis. The signal generated by thelabeled peptide is detected by a scanning device that will varyaccording to the type of label used, and the signal is then converted toan image of the site of angiogenesis.

In another embodiment, rather than directly labeling a KDR or VEGF/KDRcomplex binding polypeptide with a detectable label or radiotherapeuticconstruct, the peptide(s) of the invention can be conjugated with, forexample, avidin, biotin, or an antibody or antibody fragment that willbind the detectable label or radiotherapeutic. For example, one or moreKDR-binding peptides can be conjugated to streptavidin (potentiallygenerating multivalent binding) for in vivo binding to KDR-expressingcells. After the unbound targeting construct has cleared from the body,a biotinylated detectable label or radiotherapeutic construct (e.g., achelate molecule complexed with a radioactive metal) can be infused andwill rapidly concentrate at the site where the targeting construct isbound. This approach in some situations can reduce the time requiredafter administering the detectable label until imaging can take place.It can also increase signal to noise ratio in the target site, anddecrease the dose of the detectable label or radiotherapeutic constructrequired. This is particularly useful when a radioactive label orradiotherapeutic is used as the dose of radiation that is delivered tonormal but radiation-sensitive sites in the body, such as bone-marrow,kidneys, and liver is decreased. This approach, sometimes referred to aspre-targeting or two-step, or three-step approaches was reviewed by S.F. Rosebrough in Q. J. Nucl. Med., 40:234-251 (1996), which isincorporated by reference herein.

The present invention also includes methods for detecting, monitoringand/or evaluating therapeutic responses by imaging followingadministration of one or more KDR binding moieties described hereinconjugated to a detectable label. Such methods are especially useful formonitoring the therapeutic response of disorders related to angiogenesisand/or hyperproliferative diseases, especially diseases associated withendothelial cell hyperproliferation, such as cancer, particularlyprostate cancer, mammary cancer, ovarian cancer, liver cancer, coloncancer, renal cancer, bone cancer, bladder cancer, pancreatic cancer,lung cancer, uterine cancer and testicular cancer.

Such methods may include the steps of (a) administering a contrast agentcomprising one or more KDR-binding moieties conjugated to a detectablelabel to a subject with cancer or another disease related toangiogenesis or cell (particularly endothelial cell) hyperproliferation;(b) obtaining an initial image of the disease; (c) administering atherapeutic agent appropriate for the treatment of the disease; (d)obtaining a subsequent image of the hyperproliferative disease; and (e)comparing the initial and the subsequent images to evaluate theeffectiveness of the therapy.

In certain embodiments the subject is a mammal. In certain embodimentsthe subject is a human, and the disease is cancer. The therapeutic agentis an agent appropriate to treat a disorder associated with angiogesisor cell hyperproliferation. In one embodiment the therapeutic agent isan anti-cancer agent. Such agents are known to those skilled in the artand are discussed infra. In another embodiment, the anti-cancertreatment may include treatment of the patient with radiotherapy, RFablation or focused ultrasound.

The therapeutic response may be any type of therapeutic response knownto those of ordinary skill in the art. In certain embodiments, thetherapeutic response is reduction in tumor size and/or vascularity.Thus, the present invention provides medical personnel with the abilityto adjust treatment strategies based on the adequacy of the therapeuticresponse. If the therapeutic response is insufficient, a differenttherapeutic agent or a higher dose of the same therapeutic agent may bewarranted.

A. Magnetic Resonance Imaging (MRI)

The KDR or VEGF/KDR complex binding moieties of the present inventioncan advantageously be conjugated with one or more paramagnetic metalchelates in order to form a contrast agent for use in MRI. Preferredparamagnetic metal ions have atomic numbers 21-29, 42, 44, or 57-83.This includes ions of the transition metal or lanthanide series thathave one, and more preferably five or more, unpaired electrons and amagnetic moment of at least 1.7 Bohr magneton. Preferred paramagneticmetals include, but are not limited to, chromium (III), manganese (II),manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper(II), praseodymium (III), neodymium (III), samarium (III), gadolinium(III), terbium (III), dysprosium (III), holmium (III), erbium (III),europium (III) and ytterbium (III), chromium (III), iron (III), andgadolinium (III). The trivalent cation, Gd³⁺, is particularly preferredfor MRI contrast agents, due to its high relaxivity and low toxicity,with the further advantage that it exists in only one biologicallyaccessible oxidation state, which minimizes undesired metabolysis of themetal by a patient. Another useful metal is Cr³⁺, which is relativelyinexpensive. Gd(III) chelates have been used for clinical and radiologicMR applications since 1988, and approximately 30% of MR exams currentlyemploy a gadolinium-based contrast agent. Additionally, heteromultimersof the present invention also can be conjugated with one or moresuperparamagnetic particles.

The practitioner will select a metal according to dose required todetect angiogenesis and considering other factors such as toxicity ofthe metal to the subject (Tweedle et al., Magnetic Resonance Imaging(2nd ed.), vol. 1, Partain et al., Eds. (W.B. Saunders Co. 1988), pp.796-797). Generally, the desired dose for an individual metal will beproportional to its relaxivity, modified by the biodistribution,pharmacokinetics and metabolism of the metal.

The paramagnetic metal chelator(s) is a molecule having one or morepolar groups that act as a ligand for, and complex with, a paramagneticmetal. Suitable chelators are known in the art and include acids withmethylene phosphonic acid groups, methylene carbohydroxamine acidgroups, carboxyethylidene groups, or carboxymethylene groups. Examplesof chelators include, but are not limited to,diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclo-tetradecane-1,4,7,10-tetraacetic acid (DOTA),1-substituted 1,4,7-tricarboxymethyl-1,4,7,10-teraazacyclododecane(DO3A), ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylene bis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-Cl-EHPG, 5Br-EHPG,5-Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) andderivatives thereof; the class of macrocyclic compounds, which containat least 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (O and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM). Apreferred chelator for use in the present invention is DTPA, and the useof DO3A is particularly preferred. Examples of representative chelatorsand chelating groups contemplated by the present invention are describedin WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO97/36619, PCT/US98/01473, PCT/US98/20182, and U.S. Pat. No. 4,899,755,U.S. Pat. No. 5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No.6,143,274, all of which are hereby incorporated by reference.

In accordance with the present invention, the chelator of the MRIcontrast agent is coupled to the KDR or VEGF/KDR complex bindingpolypeptide. The positioning of the chelate(s) should be selected so asnot to interfere with the binding affinity or specificity of the KDR orVEGF/KDR complex binding polypeptide. Preferably, the chelate(s) will beappended either to the N-terminus or the C-terminus, however thechelate(s) may also be attached anywhere within the sequence. Inpreferred embodiments, a chelator having a free central carboxylic acidgroup (e.g., DTPA-Asp(β-COOH)—)OtBu) makes it easy to attach at theN-terminus of the peptide by formation of an amide bond. The chelate(s)can also be attached at the C-terminus with the aid of a linker.Alternatively, isothiocyanate conjugation chemistry can be employed as away of linking the appropriate isothiocyanate group bearing DTPA to afree amino group anywhere within the peptide sequence.

In general, the KDR or VEGF/KDR complex binding moiety can be bounddirectly or covalently to the metal chelator (or other detectablelabel), or it may be coupled or conjugated to the metal chelator using alinker, which may be, without limitation, amide, urea, acetal, ketal,double ester, carbonyl, carbamate, thiourea, sulfone, thioester, ester,ether, disulfide, lactone, imine, phosphoryl, or phosphodiesterlinkages; substituted or unsubstituted saturated or unsaturated alkylchains; linear, branched, or cyclic amino acid chains of a single aminoacid or different amino acids (e.g., extensions of the N- or C-terminusof the KDR or VEGF/KDR complex binding moiety); derivatized orunderivatized polyethylene glycol, polyoxyethylene, or polyvinylpyridinechains; substituted or unsubstituted polyamide chains; derivatized orunderivatized polyamine, polyester, polyethylenimine, polyacrylate,poly(vinyl alcohol), polyglycerol, or oligosaccharide (e.g., dextran)chains; alternating block copolymers; malonic, succinic, glutaric,adipic and pimelic acids; caproic acid; simple diamines and dialcohols;any of the other linkers disclosed herein; or any other simple polymericlinkers known in the art (see, e.g., WO 98/18497, WO 98/18496).Preferably the molecular weight of the linker can be tightly controlled.The molecular weights can range in size from less than 100 to greaterthan 1000. Preferably the molecular weight of the linker is less than100. In addition, it may be desirable to utilize a linker that isbiodegradable in vivo to provide efficient routes of excretion for theimaging reagents of the present invention. Depending on their locationwithin the linker, such biodegradable functionalities can include ester,double ester, amide, phosphoester, ether, acetal, and ketalfunctionalities.

In general, known methods can be used to couple the metal chelate(s) andthe KDR or VEGF/KDR complex binding moiety using linkers. See, e.g., WO95/28967, WO 98/18496, WO 98/18497 and discussion therein. The KDR orVEGF/KDR complex binding moiety can be linked through its N- orC-terminus via an amide bond, for example, to a metal coordinatingbackbone nitrogen of a metal chelate or to an acetate arm of the metalchelate itself. The present invention contemplates linking of thechelate on any position, provided the metal chelate retains the abilityto bind the metal tightly in order to minimize toxicity. Similarly, theKDR or VEGF/KDR complex binding moiety may be modified or elongated inorder to generate a locus for attachment to a metal chelate, providedsuch modification or elongation does not eliminate its ability to bindKDR or VEGF/KDR complex.

MRI contrast reagents prepared according to the disclosures herein maybe used in the same manner as conventional MRI contrast reagents. Whenimaging a site of angiogenesis, certain MR techniques and pulsesequences may be preferred to enhance the contrast of the site to thebackground blood and tissues. These techniques include (but are notlimited to), for example, black blood angiography sequences that seek tomake blood dark, such as fast spin echo sequences (see, e.g., Alexanderet al., Magnetic Resonance in Medicine, 40(2): 298-310 (1998)) andflow-spoiled gradient echo sequences (see, e.g., Edelman et al.,Radiology, 177(1): 45-50 (1990)). These methods also include flowindependent techniques that enhance the difference in contrast, such asinversion-recovery prepared or saturation-recovery prepared sequencesthat will increase the contrast between angiogenic tumor and backgroundtissues. Finally, magnetization transfer preparations may also improvecontrast with these agents (see, e.g., Goodrich et al., InvestigativeRadiology, 31(6): 323-32 (1996)).

The labeled reagent is administered to the patient in the form of aninjectable composition. The method of administering the MRI contrastagent is preferably parenterally, meaning intravenously,intraarterially, intrathecally, interstitially, or intracavitarilly. Forimaging active angiogenesis, intravenous or intraarterial administrationis preferred. For MRI, it is contemplated that the subject will receivea dosage of contrast agent sufficient to enhance the MR signal at thesite of angiogenesis at least 10%. After injection with the KDR orVEGF/KDR complex binding moiety-containing MRI reagent, the patient isscanned in the MRI machine to determine the location of any sites ofangiogenesis. In therapeutic settings, upon angiogenesis (e.g., tumor)localization, a tumorcidal agent or anti-angiogenic agent (e.g.,inhibitors of VEGF) can be immediately administered, if necessary, andthe patient can be subsequently scanned to visualize tumor regression orarrest of angiogenesis.

B. Ultrasound Imaging

When ultrasound is transmitted through a substance, the acousticproperties of the substance will depend upon the velocity of thetransmissions and the density of the substance. Changes in the acousticproperties will be most prominent at the interface of differentsubstances (solids, liquids, gases). Ultrasound contrast agents areintense sound wave reflectors because of the acoustic differencesbetween the agent and the surrounding tissue. Gas containing or gasgenerating ultrasound contrast agents are particularly useful because ofthe acoustic difference between liquid (e.g., blood) and thegas-containing or gas generating ultrasound contrast agent. Because oftheir size, ultrasound contrast agents comprising microbubbles,ultrasound contrast agents, and the like may remain for a longer time inthe blood stream after injection than other detectable moieties; atargeted KDR or VEGF/KDR complex-specific ultrasound agent therefore maydemonstrate superior imaging of sites of angiogenesis.

In this aspect of the invention, the KDR or VEGF/KDR complex bindingmoiety may be linked to a material that is useful for ultrasoundimaging. For example, the KDR or VEGF/KDR complex binding polypeptidesmay be linked to materials employed to form vesicles (e.g.,microbubbles, ultrasound contrast agents, microspheres, etc.), oremulsions containing a liquid or gas that functions as the detectablelabel (e.g., an echogenic gas or material capable of generating anechogenic gas). Materials for the preparation of such vesicles includesurfactants, lipids, sphingolipids, oligolipids, phospholipids,proteins, polypeptides, carbohydrates, and synthetic or naturalpolymeric materials. See, e.g., WO 98/53857, WO 98/18498, WO 98/18495,WO 98/18497, WO 98/18496, and WO 98/18501, incorporated herein byreference in their entirety.

For contrast agents comprising suspensions of stabilized microbubbles (apreferred embodiment), phospholipids, and particularly saturatedphospholipids are preferred. The preferred gas-filled microbubbles ofthe invention can be prepared by means known in the art, such as, forexample, by a method described in any one of the following patents: EP554213, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP682530, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No.6,183,725, EP 474833, U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519,U.S. Pat. No. 5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No.5,658,551, U.S. Pat. No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat.No. 6,110,443, U.S. Pat. No. 6,136,293, EP 619743, U.S. Pat. No.5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat.No. 6,187,288, and U.S. Pat. No. 5,908,610, which are incorporated byreference herein in their entirety. In a preferred embodiment, at leastone of the phospholipid moieties has the structure 18 or 19 (FIG. 33)and described in U.S. Pat. No. 5,686,060, which is herein incorporatedby reference. In ultrasound applications the contrast agents formed byphospholipid stabilized microbubbles can be administered, for example,in doses such that the amount of phospholipid injected is in the range0.1 to 200 μg/kg body weight, preferably from about 0.1 to 30 μg/kg.

Examples of suitable phospholipids include esters of glycerol with oneor two molecules of fatty acids (the same or different) and phosphoricacid, wherein the phosphoric acid residue is in turn bonded to ahydrophilic group, such as choline, serine, inositol, glycerol,ethanolamine, and the like groups. Fatty acids present in thephospholipids are in general long chain aliphatic acids, typicallycontaining from 12 to 24 carbon atoms, preferably from 14 to 22, thatmay be saturated or may contain one or more unsaturations. Examples ofsuitable fatty acids are lauric acid, myristic acid, palmitic acid,stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid,and linolenic acid. Mono esters of phospholipid are also known in theart as the “lyso” forms of the phospholipids.

Further examples of phospholipids are phosphatidic acids, i.e., thediesters of glycerol-phosphoric acid with fatty acids, sphingomyelins,i.e., those phosphatidylcholine analogs where the residue of glyceroldiester with fatty acids is replaced by a ceramide chain, cardiolipins,i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid,gangliosides, cerebrosides, etc. As used herein, the term phospholipidsincludes either naturally occurring, semisynthetic or syntheticallyprepared products that can be employed either singularly or as mixtures.Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins.

Examples of synthetic phospholipids are e.g.,dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”),diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine(“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatid-ylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts,distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts,distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid(“DAPA”) and its alkali metal salts, dimyristoylphosphatidylethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine(“DPPE”), distearoyl phosphatidyl-ethanolamine (“DSPE”), dimyristoylphosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”),dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin(“DPSP”), and distearoyl sphingomyelin (“DSSP”).

Other preferred phospholipids include dipalmitoylphosphatidylcholine,dipalmitoylphosphatidic acid and dipalmitoylphosphatidylserine. Thecompositions also may contain PEG-4000 and/or palmitic acid. Any of thegases disclosed herein or known to the skilled artisan may be employed;however, inert gases, such as SF₆ or fluorocarbons like CF₄, C₃F₈ andC₄F₁₀, are preferred.

The preferred microbubble suspensions of the present invention may beprepared from phospholipids using known processes such as afreeze-drying or spray-drying solutions of the crude phospholipids in asuitable solvent or using the processes set forth in EP 554213; U.S.Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S.Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No.5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat.No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S.Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No.5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S.Pat. No. 5,908,610, which are incorporated by reference herein in theirentirety. Most preferably, the phospholipids are dissolved in an organicsolvent and the solution is dried without going through a liposomeformation stage. This can be done by dissolving the phospholipids in asuitable organic solvent together with a hydrophilic stabilizersubstance or a compound soluble both in the organic solvent and waterand freeze-drying or spray-drying the solution. In this embodiment thecriteria used for selection of the hydrophilic stabilizer is itssolubility in the organic solvent of choice. Examples of hydrophilicstabilizer compounds soluble in water and the organic solvent are, e.g.,a polymer, like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA),polyethylene glycol (PEG), etc., malic acid, glycolic acid, maltol, andthe like. Such hydrophilic compounds also aid in homogenizing themicrobubbles size distribution and enhance stability under storage. Anysuitable organic solvent may be used as long as its boiling point issufficiently low and its melting point is sufficiently high tofacilitate subsequent drying. Typical organic solvents include, forexample, dioxane, cyclohexanol, tertiary butanol, tetrachlorodifluoroethylene (C₂Cl₄F₂) or 2-methyl-2-butanol. 2-methyl-2-butanol and C₂Cl₄F₂are preferred.

Prior to formation of the suspension of microbubbles by dispersion in anaqueous carrier, the freeze dried or spray dried phospholipid powdersare contacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments that will stabilize themicrobubbles of the gas dispersed therein. This method permitsproduction of suspensions of microbubbles that are stable even whenstored for prolonged periods and are obtained by simple dissolution ofthe dried laminarized phospholipids (which have been stored under adesired gas) without shaking or any violent agitation.

Alternatively, microbubbles can be prepared by suspending a gas into anaqueous solution at high agitation speed, as disclosed e.g. in WO97/29783. A further process for preparing microbubbles is disclosed inco-pending European patent application no. 03002373, herein incorporatedby reference, which comprises preparing an emulsion of an organicsolvent in an aqueous medium in the presence of a phospholipid andsubsequently lyophilizing said emulsion, after optional washing and/orfiltration steps.

Additives known to those of ordinary skill in the art can be included inthe suspensions of stabilized microbubbles. For instance, non-filmforming surfactants, including polyoxypropylene glycol andpolyoxyethylene glycol and similar compounds, as well as variouscopolymers thereof; fatty acids such as myristic acid, palmitic acid,stearic acid, arachidic acid or their derivatives, ergosterol,phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate,ascorbyl palmitate and butylated hydroxytoluene may be added. The amountof these non-film forming surfactants is usually up to 50% by weight ofthe total amount of surfactants but preferably between 0 and 30%.

Other gas containing suspensions include those disclosed in, forexample, U.S. Pat. No. 5,798,091, WO 97/29783, also EP 881 915,incorporated herein by reference in their entirety. These agents may beprepared as described in U.S. Pat. No. 5,798,091 or WO97/29783.

Another preferred ultrasound contrast agent comprises ultrasoundcontrast agents. The term “microballoon” refers to gas filled bodieswith a material boundary or envelope. More on microballoon formulationsand methods of preparation may be found in EP 324 938 (U.S. Pat. No.4,844,882); U.S. Pat. No. 5,711,933; U.S. Pat. No. 5,840,275; U.S. Pat.No. 5,863,520; U.S. Pat. No. 6,123,922; U.S. Pat. No. 6,200,548; U.S.Pat. No. 4,900,540; U.S. Pat. No. 5,123,414; U.S. Pat. No. 5,230,882;U.S. Pat. No. 5,469,854; U.S. Pat. No. 5,585,112; U.S. Pat. No.4,718,433; U.S. Pat. No. 4,774,958; WO 95/01187; U.S. Pat. No.5,529,766; U.S. Pat. No. 5,536,490; and U.S. Pat. No. 5,990,263, thecontents of which are incorporated herein by reference.

The preferred microballoons have an envelope including a biodegradablephysiologically compatible polymer or, a biodegradable solid lipid. Thepolymers useful for the preparation of the microballoons of the presentinvention can be selected from the biodegradable physiologicallycompatible polymers, such as any of those described in any of thefollowing patents: EP 458745, U.S. Pat. No. 5,711,933, U.S. Pat. No.5,840,275, EP 554213, U.S. Pat. No. 5,413,774 and U.S. Pat. No.5,578,292, the entire contents of which are incorporated herein byreference. In particular, the polymer can be selected from biodegradablephysiologically compatible polymers, such as polysaccharides of lowwater solubility, polylactides and polyglycolides and their copolymers,copolymers of lactides and lactones such as ε-caprolactone,γ-valerolactone and polypeptides. Other suitable polymers includepoly(ortho)esters (see e.g., U.S. Pat. No. 4,093,709; U.S. Pat. No.4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646); polylacticand polyglycolic acid and their copolymers, for instance DEXON (see J.Heller, Biomaterials 1 (1980), 51; poly(DL-lactide-co-γ-caprolactone),poly(DL-lactide-co-γ-valerolactone),poly(DL-lactide-co-γ-butyrolactone), polyalkylcyano-acrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones (A.S. Angeloni, P. Ferruti, M. Tramontini and M. Casolaro, The Mannichbases in polymer synthesis: 3. Reduction of poly(beta-aminoketone)s topoly(gamma-aminoalcohol)s and their N-alkylation topoly(gamma-hydroxyquaternary ammonium salt)s, Polymer 23, pp 1693-1697,1982.); polyphosphazenes (Allcock, Harry R. Polyphosphazenes: newpolymers with inorganic backbone atoms (Science 193:1214-19 (1976)) andpolyanhydrides. The microballoons of the present invention can also beprepared according to the methods of WO-A-96/15815, incorporated hereinby reference, where the microballoons are made from a biodegradablemembrane comprising biodegradable lipids, preferably selected from mono-di-, tri-glycerides, fatty acids, sterols, waxes and mixtures thereof.Preferred lipids are di- or tri-glycerides, e.g., di- or tri-myristin,-palmityn or -stearin, in particular tripalmitin or tristearin. Themicroballoons may employ any of the gases disclosed herein of known tothe skilled artisan; however, inert gases such as fluorinated gases arepreferred. The microballoons may be suspended in a pharmaceuticallyacceptable liquid carrier with optional additives known to those ofordinary skill in the art and stabilizers.

Other gas-containing contrast agent formulations include microparticles(especially aggregates of microparticles) having gas contained thereinor otherwise associated therewith (for example being adsorbed on thesurface thereof and/or contained within voids, cavities or porestherein). Methods for the preparation of these agents are as describedin EP 0122624; EP 0123235; EP 0365467; U.S. Pat. No. 5,558,857; U.S.Pat. No. 5,607,661; U.S. Pat. No. 5,637,289; U.S. Pat. No. 5,558,856;U.S. Pat. No. 5,137,928; WO 95/21631 or WO 93/13809, incorporated hereinby reference in their entirety.

Any of these ultrasound compositions should also be, as far as possible,isotonic with blood. Hence, before injection, small amounts of isotonicagents may be added to any of above ultrasound contrast agentsuspensions. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2.6% glycerol solution, 5% dextrose solution, etc. Additionally, theultrasound compositions may include standard pharmaceutically acceptableadditives, including, for example, emulsifying agents, viscositymodifiers, cryoprotectants, lyoprotectants, bulking agents etc.

Any biocompatible gas may be used in the ultrasound contrast agentsuseful in the invention. The term “gas” as used herein includes anysubstances (including mixtures) substantially in gaseous form at thenormal human body temperature. The gas may thus include, for example,air, nitrogen, oxygen, CO₂, argon, xenon or krypton, fluorinated gases(including for example, perfluorocarbons, SF₆, SeF₆) a low molecularweight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), forexample, an alkane such as methane, ethane, a propane, a butane or apentane, a cycloalkane such as cyclopropane, cyclobutane orcyclopentene, an alkene such as ethylene, propene, propadiene or abutene, or an alkyne such as acetylene or propyne and/or mixturesthereof. However, fluorinated gases are preferred. Fluorinated gasesinclude materials that contain at least one fluorine atom such as SF₆,freons (organic compounds containing one or more carbon atoms andfluorine, i.e., CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀, CBrF₃, CCI₂F₂, C₂CIF₅, andCBrClF₂) and perfluorocarbons. The term perfluorocarbon refers tocompounds containing only carbon and fluorine atoms and includes, inparticular, saturated, unsaturated, and cyclic perfluorocarbons. Thesaturated perfluorocarbons, which are usually preferred, have theformula C_(n)F_(n+2), where n is from 1 to 12, preferably from 2 to 10,most preferably from 3 to 8 and even more preferably from 3 to 6.Suitable perfluorocarbons include, for example, CF₄, C₂F₆, C₃F₈ C₄F₈,C₄F₁₀, C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, and C₉F₂₀. Most preferably the gas orgas mixture comprises SF₆ or a perfluorocarbon selected from the groupconsisting of C₃F₈ C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, with C₄F₁₀being particularly preferred. See also WO 97/29783, WO 98/53857, WO98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO98/05364, WO 98/17324.

In certain circumstances it may be desirable to include a precursor to agaseous substance (e.g., a material that is capable of being convertedto a gas in vivo, often referred to as a “gas precursor”). Preferablythe gas precursor and the gas it produces are physiologicallyacceptable. The gas precursor may be pH-activated, photo-activated,temperature activated, etc. For example, certain perfluorocarbons may beused as temperature activated gas precursors. These perfluorocarbons,such as perfluoropentane, have a liquid/gas phase transition temperatureabove room temperature (or the temperature at which the agents areproduced and/or stored) but below body temperature; thus they undergo aphase shift and are converted to a gas within the human body.

As discussed, the gas can comprise a mixture of gases. The followingcombinations are particularly preferred gas mixtures: a mixture of gases(A) and (B) in which, at least one of the gases (B), present in anamount of between 0.5-41% by vol., has a molecular weight greater than80 daltons and is a fluorinated gas and (A) is selected from the groupconsisting of air, oxygen, nitrogen, carbon dioxide and mixturesthereof, the balance of the mixture being gas A.

Since ultrasound vesicles may be larger than the other detectable labelsdescribed herein, they may be linked or conjugated to a plurality of KDRor VEGF/KDR complex binding polypeptides in order to increase thetargeting efficiency of the agent. Attachment to the ultrasound contrastagents described above (or known to those skilled in the art) may be viadirect covalent bond between the KDR or VEGF/KDR complex bindingpolypeptide and the material used to make the vesicle or via a linker,as described previously. For example, see WO 98/53857 generally for adescription of the attachment of a peptide to a bifunctional PEG linker,which is then reacted with a liposome composition. See also, Lanza etal., Ultrasound in Med. & Bio., 23(6):863-870 (1997).

A number of methods may be used to prepare suspensions of microbubblesconjugated to KDR or VEGF/KDR complex binding polypeptides. For example,one may prepare maleimide-derivatized microbubbles by incorporating 5%(w/w) of N-MPB-PE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido-phenylbutyramide), (Avanti Polar-Lipids, Inc) in the phospholipid formulation.Then, solutions of mercaptoacetylated KDR-binding peptides (10 mg/mL inDMF), which have been incubated in deacetylation solution (50 mM sodiumphosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) are added to themaleimide-activated microbubble suspension. After incubation in thedark, under gentle agitation, the peptide conjugated microbubbles may bepurified by centrifugation.

Compounds that can be used for derivatization of microbubbles typicallyinclude the following components: (a) a hydrophobic portion, compatiblewith the material forming the envelope of the microbubble or of themicroballoon, in order to allow an effective incorporation of thecompound in the envelope of the vesicel; said portion is representedtypically by a lipid moiety (dipalmitin, distearoyl); and (b) a spacer(typically PEGs of different molecular weights), which may be optionalin some cases (for example, microbubbles may for instance presentdifficulties to be freeze dried if the spacer is too long) or preferredin some others (e.g., peptides may be less active when conjugated to amicroballoon with short spacers); and (c) a reactive group capable ofreacting with a corresponding reacting moiety on the peptide to beconjugated (e.g., maleimido with the —SH group of cysteine).

Alternatively, KDR-binding polypeptide conjugated microbubbles may beprepared using biotin/avidin. For example, avidin-conjugatedmicrobubbles may be prepared using a maleimide-activated phospholipidmicrobubble suspension, prepared as described above, which is added tomercaptoacetylated-avidin (which has been incubated with deacetylationsolution). Biotinylated KDR or VEGF/KDR complex-binding peptides(prepared as described herein) are then added to the suspension ofavidin-conjugated microbubbles, yielding a suspension of microbubblesconjugated to KDR or VEGF/KDR complex-binding peptides.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilized residue may be stored andtransported without need of temperature control of its environment andin particular it may be supplied to hospitals and physicians for on siteformulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities. Preferably insuch a case it can be supplied in the form of a two-component kit, whichcan include two separate containers or a dual-chamber container. In theformer case preferably the container is a conventional septum-sealedvial, wherein the vial containing the lyophilized residue of step b) issealed with a septum through which the carrier liquid may be injectedusing an optionally prefilled syringe. In such a case the syringe usedas the container of the second component is also used then for injectingthe contrast agent. In the latter case, preferably the dual-chambercontainer is a dual-chamber syringe and once the lyophilizate has beenreconstituted and then suitably mixed or gently shaken, the containercan be used directly for injecting the contrast agent. In both casesmeans for directing or permitting application of sufficient bubbleforming energy into the contents of the container are provided.

However, as noted above, in the stabilised contrast agents according tothe invention the size of the gas microbubbles is substantiallyindependent of the amount of agitation energy applied to thereconstituted dried product. Accordingly, no more than gentle handshaking is generally required to give reproducible products withconsistent microbubble size.

It can be appreciated by one of ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g. a targeting ligand to belinked to the phospholipid during reconstitution), it can be packagedwith the other components of the kit, preferably in a form or containeradapted to facilitate ready combination with the other components of thekit.

No specific containers, vial or connection systems are required; thepresent invention may use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirable substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.As to the stopper, it may be a compound or multicomponent formulationbased on an elastomer, such as poly(isobutylene) or butyl rubber.

Ultrasound imaging techniques that can be used in accordance with thepresent invention include known techniques, such as color Doppler, powerDoppler, Doppler amplitude, stimulated acoustic imaging, and two- orthree-dimensional imaging techniques. Preferably, the ultrasound imagingis effected in a contrast-specific imaging mode so as to substantiallyremove, or at least reduce, the dominant (linear) contribution of tissuein the echo signals, with respect to the (non-linear) contribution ofthe contrast agent; examples of contrast-specific imaging modes includeharmonic imaging (HI), pulse inversion (PI), power modulation (PM) andcontrast pulse sequencing (CPS) techniques, as described, for example,in “Rafter et al., Imaging technologies and techniques, CardiologyClinics 22 (2004), pp. 181-197” (the entire disclosure of which isherewith incorporated by reference).

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles may, for example, be administered in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.Microballoons-containing contrast agents are typically administered indoses such that the amount of wall-forming polymer or lipid is fromabout 10 μg/kg to about 20 mg/kg of body weight.

As shown in the Examples, ultrasound contrast agents conjugated to KDRbinding moieties of the invention, such as, for example, thosecomprising SEQ ID NOS: 356, 294 and 480 and the dimer D23, are able tobind to KDR-expressing tissue and thus are useful in providing an imageof such tissue. Indeed, compounds of the invention, such as phospholipidstabilized microbubbles conjugated to the heterodimer D23, can be usedto image angiogenic tissue in vivo.

The present invention also includes methods for detecting, monitoringand/or evaluating therapeutic responses following administration of anultrasound contrast agent conjugated to KDR binding moieties describedherein. Such methods are especially useful for monitoring thetherapeutic response of disorders related angiogenesis and/orhyperproliferative diseases, especially diseases associated withendothelial cell hyperproliferation. The disease may includeinflammatory diseases, such as, for instance, rheumatoid diseases andinflammatory bowel diseases, and cancers such as prostate cancer,mammary cancer, ovarian cancer, liver cancer, colon cancer, renalcancer, bone cancer, bladder cancer, pancreatic cancer, lung cancer,uterine cancer and testicular cancer.

Such methods may include the steps of (a) administering an ultrasoundcontrast agent described above to a subject with cancer or anotherdisease related to angiogesis or endothelial cell hyper proliferation;(b) obtaining an initial image of the disease; (c) administering atherapeutic agent appropriate for the treatment of the disease; (d)obtaining a subsequent image of the hyperproliferative disease; and (e)comparing the initial and the subsequent images to evaluate theeffectiveness of the therapy.

In certain embodiments the ultrasound contrast agent comprises a dimer,such as D5. In certain embodiments the subject is a mammal. In certainembodiments the subject is a human, and the disease is cancer. In apreferred embodiment the disease is prostate cancer.

The therapeutic agent is an agent appropriate to treat the disorder. Fordisorders associated with angiogesis or (endothelial) cellhyperproliferation, the therapeutic agent is an anti-angiogenesis agentor an anti-hyperproliferation agent. In one embodiment the therapeuticagent is an anti-cancer agent. Such agents are known to those skilled inthe art and include, for example, sunitinib, the therapeutic agentsdiscussed infra, imatinib, sorafenib and bevacizumab.

The therapeutic response may be any type of therapeutic response knownto those of ordinary skill in the art. In certain embodiments, thetherapeutic response is reduction (or increase) in tumor size and/orvascularity. Tumor size is typically assessed by visual observation ofthe image (or sequence of images) obtained by subjecting the patient toa suitable imaging technique, in particular ultrasound imaging (e.g.fundamental B-mode ultrasound imaging). Vascularity can be assessed by,for example, analyzing the time-intensity curves of ultrasound signal,to determine suitable quantification parameters (in particularindicative of the vascularization) such as Imax (maximal peakenhancement), AUC (area under the curve), WIR (Wash-in-rate) or TTP(Time-to Peak); KDR expression can instead be assessed by LPO (latephase opacification). Thus, the present invention provides medicalpersonnel with the ability to assess and if necessary adjust treatmentstrategies based on the adequacy of the therapeutic response. If thetherapeutic response is insufficient, a different therapeutic agent or ahigher dose of the same therapeutic agent may be warranted. If thetherapeutic response is sufficient the current protocol will proceedunadjusted. Evaluation of the therapeutic response following adjustmentsto the treatment protocol would proceed in the same manner.

C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging

In accordance with the present invention, a number of optical parametersmay be employed to determine the location of KDR or VEGF/KDR complexwith in vivo light imaging after injection of the subject with anoptically-labeled KDR or VEGF/KDR complex binding polypeptide. Opticalparameters to be detected in the preparation of an image may includetransmitted radiation, absorption, fluorescent or phosphorescentemission, light reflection, changes in absorbance amplitude or maxima,and elastically scattered radiation. For example, biological tissue isrelatively translucent to light in the near infrared (NIR) wavelengthrange of 650-1000 nm. NIR radiation can penetrate tissue up to severalcentimeters, permitting the use of the KDR or VEGF/KDR complex bindingpolypeptides of the present invention for optical imaging of KDR orVEGF/KDR complex in vivo.

The KDR or VEGF/KDR complex binding polypeptides may be conjugated withphotolabels, such as optical dyes, including organic chromophores orfluorophores, having extensive delocalized ring systems and havingabsorption or emission maxima in the range of 400-1500 nm. The KDR orVEGF/KDR complex binding polypeptide may alternatively be derivatizedwith a bioluminescent molecule. The preferred range of absorption maximafor photolabels is between 600 and 1000 nm to minimize interference withthe signal from hemoglobin. Preferably, photoabsorption labels havelarge molar absorptivities, e.g., >10⁵ cm⁻¹M⁻¹, while fluorescentoptical dyes will have high quantum yields. Examples of optical dyesinclude, but are not limited to those described in WO 98/18497, WO98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO97/18841, WO 96/23524, WO 98/47538, and references cited therein. Thephotolabels may be covalently linked directly to the KDR or VEGF/KDRcomplex binding peptide or linked to the KDR or VEGF/KDR complex bindingpeptide via a linker, as described previously.

After injection of the optically-labeled KDR or VEGF/KDR complex bindingmoiety, the patient is scanned with one or more light sources (e.g., alaser) in the wavelength range appropriate for the photolabel employedin the agent. The light used may be monochromatic or polychromatic andcontinuous or pulsed. Transmitted, scattered, or reflected light isdetected via a photodetector tuned to one or multiple wavelengths todetermine the location of KDR or VEGF/KDR complex in the subject.Changes in the optical parameter may be monitored over time to detectaccumulation of the optically-labeled reagent at the site ofangiogenesis. Standard image processing and detecting devices may beused in conjunction with the optical imaging reagents of the presentinvention.

The optical imaging reagents described above may also be used foracousto-optical or sonoluminescent imaging performed withoptically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO98/57666, and references cited therein). In acousto-optical imaging,ultrasound radiation is applied to the subject and affects the opticalparameters of the transmitted, emitted, or reflected light. Insonoluminescent imaging, the applied ultrasound actually generates thelight detected. Suitable imaging methods using such techniques aredescribed in WO 98/57666.

D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy

The KDR or VEGF/KDR complex binding moieties may be conjugated with aradionuclide reporter appropriate for scintigraphy, SPECT, or PETimaging and/or with a radionuclide appropriate for radiotherapy.Constructs in which the KDR or VEGF/KDR complex binding moieties areconjugated with both a chelator for a radionuclide useful for diagnosticimaging and a chelator useful for radiotherapy are within the scope ofthe invention.

For use as a PET agent a peptide is complexed with one of the variouspositron emitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As,^(94m)Tc, or ¹¹⁰In. The binding moieties of the invention can also belabeled by halogenation using radionuclides such as ¹⁸F, ¹²⁴I, ¹²⁵I,¹³¹I, ¹²³I, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclides forscintigraphy or radiotherapy include ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc,⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho,¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi,²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Auand ¹⁹⁹Au. The choice of metal will be determined based on the desiredtherapeutic or diagnostic application. For example, for diagnosticpurposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,and ¹¹¹In. For therapeutic purposes, the preferred radionuclides include⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁷⁵Yb, ¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au. ^(99m)Tc is particularlypreferred for diagnostic applications because of its low cost,availability, imaging properties, and high specific activity. Thenuclear and radioactive properties of Tc-99m make this isotope an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹Mo—^(99m)Tc generator.

The metal radionuclides may be chelated by, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S.Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556,U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelatorsknown in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA,DO3A, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. Pat.No. 5,720,934). For example, N₄ chelators are described in U.S. Pat. No.6,143,274; U.S. Pat. No. 6,093,382; U.S. Pat. No. 5,608,110; U.S. Pat.No. 5,665,329; U.S. Pat. No. 5,656,254; and U.S. Pat. No. 5,688,487.Certain N₃S chelators are described in PCT/CA94/00395, PCT/CA94/00479,PCT/CA95/00249 and in U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,976,495;and U.S. Pat. No. 5,780,006. The chelator may also include derivativesof the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3),which contains an N₃S, and N₂S₂ systems such as MAMA(monoamidemonoaminedithiols), DADS (N₂S diaminedithiols), CODADS and thelike. These ligand systems and a variety of others are described in Liuand Edwards, Chem Rev., 99:2235-2268 (1999) and references therein.

The chelator may also include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. No. 5,183,653; U.S. Pat. No. 5,387,409; and U.S.Pat. No. 5,118,797, the disclosures of which are incorporated byreference herein, in their entirety.

In another embodiment, disulfide bonds of a KDR or VEGF/KDR complexbinding polypeptide of the invention are used as two ligands forchelation of a radionuclide such as ^(99m)Tc. In this way the peptideloop is expanded by the introduction of Tc (peptide-S-S-peptide changedto peptide-S-Tc-S-peptide). This has also been used in other disulfidecontaining peptides in the literature (Chen et al., J. Nucl. Med.,42:1847-1855 (2001)) while maintaining biological activity. The otherchelating groups for Tc can be supplied by amide nitrogens of thebackbone, another cystine amino acid or other modifications of aminoacids.

Particularly preferred metal chelators include those of Formula 20, 21,22, 23a, 23b, 24a, 24b and 25 (FIGS. 34A-F) and FIG. 35. Formulas 20-22(FIGS. 34A-C) are particularly useful for lanthanides such asparamagnetic Gd³⁺ and radioactive lanthanides such as ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm,¹¹¹In, or ¹⁶⁶Ho. Formulas 23a-24b (FIGS. 34D and F) and FIG. 35 areparticularly useful for radionuclides ^(99m)Tc, ¹⁸⁶Re, or ¹⁸⁸Re. Formula25 (FIG. 34F) and the structure shown in FIG. 35 are particularly usefulfor ^(99m)Tc. These and other metal chelating groups are described inU.S. Pat. No. 6,093,382 and U.S. Pat. No. 5,608,110, which areincorporated by reference herein in their entirety. Additionally, thechelating group of formula 22 (FIG. 34C) is described in, for example,U.S. Pat. No. 6,143,274; the chelating group of formula 24 is describedin, for example, U.S. Pat. No. 5,627,286 and U.S. Pat. No. 6,093,382,and the chelating groups of formula 25 and FIG. 35 are described in, forexample, U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,780,006; and U.S. Pat.No. 5,976,495.

In the above Formulas 24a and 24b (FIG. 34E), X is either CH₂ or O; Y isC₁-C₁₀ branched or unbranched alky, aryl, aryloxy, arylamino,arylaminoacyl, or arylalkyl comprising C₁-C₁₀ branched or unbranchedalkyl groups, hydroxy or C₁-C₁₀ branched or unbranched polyhydroxyalkylgroups, C₁-C₁₀ branched or unbranched hydroxy or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups; J is C(═O)—, OC(═O)—, SO₂—, NC(═O)—,NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—, homopolyamides orheteropolyamines derived from synthetic or naturally occurring aminoacids; and n is 1-100. Other variants of these structures are described,for example, in U.S. Pat. No. 6,093,382. The disclosures of each of theforegoing patents, applications and references are incorporated byreference herein, in their entirety.

The chelators may be covalently linked directly to the KDR or VEGF/KDRcomplex binding moiety or linked to the KDR or VEGF/KDR complex bindingpolypeptide via a linker, as described previously, and then directlylabeled with the radioactive metal of choice (see, WO 98/52618, U.S.Pat. No. 5,879,658, and U.S. Pat. No. 5,849,261).

Complexes of radioactive technetium are particularly useful fordiagnostic imaging and complexes of radioactive rhenium are particularlyuseful for radiotherapy. In forming a complex of radioactive technetiumwith the reagents of this invention, the technetium complex, preferablya salt of Tc-99m pertechnetate, is reacted with the reagent in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions; the most preferred reducing agent is stannouschloride. Means for preparing such complexes are conveniently providedin a kit form comprising a sealed vial containing a predeterminedquantity of a reagent of the invention to be labeled and a sufficientamount of reducing agent to label the reagent with Tc-99m.Alternatively, the complex may be formed by reacting a peptide of thisinvention conjugated with an appropriate chelator with a pre-formedlabile complex of technetium and another compound known as a transferligand. This process is known as ligand exchange and is well known tothose skilled in the art. The labile complex may be formed using suchtransfer ligands as tartrate, citrate, gluconate or mannitol, forexample. Among the Tc-99m pertechnetate salts useful with the presentinvention are included the alkali metal salts such as the sodium salt,or ammonium salts or lower alkyl ammonium salts.

Preparation of the complexes of the present invention where the metal isradioactive rhenium may be accomplished using rhenium starting materialsin the +5 or +7 oxidation state. Examples of compounds in which rheniumis in the Re(VII) state are NH₄ReO₄ or KReO₄. Re(V) is available as, forexample, [ReOCl₄](NBu₄), [ReOCl₄](AsPh₄), ReOCl₃(PPh₃)₂ and asReO₂(pyridine)₄ ⁺, where Ph is phenyl and Bu is n-butyl. Other rheniumreagents capable of forming a rhenium complex may also be used.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 mCi to 100 mCi permL.

Generally, the unit dose to be administered has a radioactivity of about0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution tobe injected at unit dosage is from about 0.01 mL to about 10 mL.

Typical doses of a radionuclide-labeled KDR or VEGF/KDR complex bindingimaging agents according to the invention provide 10-20 mCi. Afterinjection of the KDR or VEGF/KDR complex-specific radionuclide imagingagent into the patient, a gamma camera calibrated for the gamma rayenergy of the nuclide incorporated in the imaging agent is used to imageareas of uptake of the agent and quantify the amount of radioactivitypresent in the site.

Imaging of the site in vivo can take place in a matter of a few minutes.However, imaging can take place, if desired, hours or even longer, afterthe radiolabeled peptide is injected into a patient. In most instances,a sufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 of an hour to permit the taking ofscintiphotos.

Proper dose schedules for the radiotherapeutic compounds of the presentinvention are known to those skilled in the art. The compounds can beadministered using many methods that include, but are not limited to, asingle or multiple IV or IP injections, using a quantity ofradioactivity that is sufficient to cause damage or ablation of thetargeted KDR-expressing tissue, but not so much that substantive damageis caused to non-target (normal tissue). The quantity and dose requiredis different for different constructs, depending on the energy andhalf-life of the isotope used, the degree of uptake and clearance of theagent from the body and the mass of the tumor. In general, doses canrange from a single dose of about 30-50 mCi to a cumulative dose of upto about 3 Curies.

The radiotherapeutic compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and may include, for example, para-aminobenzoic acid, ascorbic acid,gentistic acid and the like.

A single or multi-vial kit that contains all of the components needed toprepare the complexes of this invention, other than the radionuclide, isan integral part of this invention.

A single-vial kit preferably contains a chelating ligand, a source ofstannous salt, or other pharmaceutically acceptable reducing agent, andis appropriately buffered with pharmaceutically acceptable acid or baseto adjust the pH to a value of about 3 to about 9. The quantity and typeof reducing agent used would depend highly on the nature of the exchangecomplex to be formed. The proper conditions are well known to those thatare skilled in the art. It is preferred that the kit contents be inlyophilized form. Such a single vial kit may optionally contain labileor exchange ligands such as glucoheptonate, gluconate, mannitol, malate,citric or tartaric acid and can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or α, β, or γ cyclodextrin that serve to improve theradiochemical purity and stability of the final product. The kit mayalso contain stabilizers, bulking agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial may contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g., the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains theligand, as well as buffers appropriate to adjust the pH to its optimalvalue. After a reaction time of about 5 to 60 minutes, the complexes ofthe present invention are formed. It is advantageous that the contentsof both vials of this multi-vial kit be lyophilized. As above, reactionmodifiers, exchange ligands, stabilizers, bulking agents, etc. may bepresent in either or both vials.

As shown in the Examples, compounds of the invention comprising aradionuclide, particularly heteromultimers such as D10 conjugated to aradionuclide (optionally via a chelator), are useful in imaging KDR orVEGF/KDR complex expressing tissue (such as angiogenic tissue).

Additionally, the Examples establish that compounds of the inventionconjugated to a therapeutic radionuclide, particularly heteromultimerssuch as D13 conjugated to a chelator and complexed with a therapeuticradionuclide, are useful in radiotherapy of tumors expressing KDR.

Other Therapeutic Applications

The KDR or VEGF/KDR complex binding polypeptides of the presentinvention can be used to improve the activity of therapeutic agents suchas anti-angiogenic or tumorcidal agents against undesired angiogenesissuch as occurs in neoplastic tumors, by providing or improving theiraffinity for KDR or VEGF/KDR complex and their residence time at a KDRor VEGF/KDR complex on endothelium undergoing angiogenesis. In thisaspect of the invention, hybrid agents are provided by conjugating a KDRor VEGF/KDR complex binding polypeptide according to the invention witha therapeutic agent. The therapeutic agent may be a radiotherapeutic,discussed above, a drug, chemotherapeutic or tumorcidal agent, geneticmaterial or a gene delivery vehicle, etc. The KDR or VEGF/KDR complexbinding polypeptide portion of the conjugate causes the therapeutic to“home” to the sites of KDR or VEGF/KDR complex (i.e., activatedendothelium), and to improve the affinity of the conjugate for theendothelium, so that the therapeutic activity of the conjugate is morelocalized and concentrated at the sites of angiogenesis. Such conjugateswill be useful in treating angiogenesis-associated diseases, especiallyneoplastic tumor growth and metastasis, in mammals, including humans,which method comprises administering to a mammal in need thereof aneffective amount of a KDR or VEGF/KDR complex binding polypeptideaccording to the invention conjugated with a therapeutic agent. Theinvention also provides the use of such conjugates in the manufacture ofa medicament for the treatment of angiogenesis associated diseases inmammals, including humans.

Suitable therapeutic agents for use in this aspect of the inventioninclude, but are not limited to: antineoplastic agents, such as platinumcompounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside,arabinosyl adenine, mercaptopolylysine, vincristine, busulfan,chlorambucil, melphalan (e.g., PAM, L-PAM, or phenylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin(actinomycin D), daunorubcin hydrochloride, doxorubicin hydrochloride,taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testoiactone, trilostane, amsacrine(m-AMSA), aparaginase (L-aparaginase), Erwina aparaginase, etoposide(VP-16), interferon cx-2a, Interferon cx-2b, teniposide (VM-26,vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate,adriamycin, and arabinosyl; anti-angiogenic agents such as tyrosinekinase inhibitors with activity toward signaling molecules important inangiogenesis and/or tumor growth such as SU5416 and SU6668(Sugen/Pharmacia & Upjohn), endostatin (EntreMed), angiostatin(EntreMed), Combrestatin (Oxigene), cyclosporine, 5-fluorouracil,vinblastine, doxorubicin, paclitaxel, daunorubcin, immunotoxins;coagulation factors; antivirals such as acyclovir, amantadineazidothymidine (AZT or Zidovudine), ribavirin and vidarabine monohydrate(adenine arahinoside, ara-A); antibiotics, antimalarials, antiprotozoanssuch as chloroquine, hydroxychloroquine, metroidazole, quinine andmeglumine antimonate; anti-inflammatories such as diflunisal, ibuprofen,indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates.

The KDR or VEGF/KDR complex binding polypeptides of the presentinvention may also be used to target genetic material to KDR-expressingcells. Thus, they may be useful in gene therapy, particularly fortreatment of diseases associated with angiogenesis. In this embodiment,genetic material or one or more delivery vehicles containing geneticmaterial useful in treating an angiogenesis-related disease may beconjugated to one or more KDR binding moieties of the invention andadministered to a patient. The genetic material may include nucleicacids, such as RNA or DNA, of either natural or synthetic origin,including recombinant RNA and DNA and antisense RNA and DNA. Types ofgenetic material that may be used include, for example, genes carried onexpression vectors such as plasmids, phagemids, cosmids, yeastartificial chromosomes (YAC's) and defective or “helper” viruses,antigene nucleic acids, both single and double stranded RNA and DNA andanalogs thereof, such as phosphorothioate and phosphorodithioateoligodeoxynucleotides. Additionally, the genetic material may becombined, for example, with lipids, proteins or other polymers. Deliveryvehicles for genetic material may include, for example, a virusparticle, a retroviral or other gene therapy vector, a liposome, acomplex of lipids (especially cationic lipids) and genetic material, acomplex of dextran derivatives and genetic material, etc.

In a preferred embodiment the constructs of the invention are utilizedin gene therapy for treatment of diseases associated with angiogenesis.In this embodiment, genetic material, or one or more delivery vehiclescontaining genetic material, e.g., useful in treating anangiogenesis-related disease, can be conjugated to one or more KDR orVEGF/KDR complex binding polypeptides or multimers (e.g., homomultimersor heteromultimers) of the invention and administered to a patient.

Constructs including genetic material and the KDR-binding polypeptidesof the invention may be used, in particular, to selectively introducegenes into angiogenic endothelial cells, which may be useful not only totreat cancer, but also after angioplasty, where inhibition ofangiogenesis may inhibit restenosis.

Therapeutic agents and the KDR or VEGF/KDR complex binding moieties ofthe invention can be linked or fused in known ways, using the same typeof linkers discussed elsewhere in this application. Preferred linkerswill be substituted or unsubstituted alkyl chains, amino acid chains,polyethylene glycol chains, and other simple polymeric linkers known inthe art. More preferably, if the therapeutic agent is itself a protein,for which the encoding DNA sequence is known, the therapeutic proteinand KDR or VEGF/KDR complex binding polypeptide may be coexpressed fromthe same synthetic gene, created using recombinant DNA techniques, asdescribed above. The coding sequence for the KDR or VEGF/KDR complexbinding polypeptide may be fused in frame with that of the therapeuticprotein, such that the peptide is expressed at the amino- orcarboxy-terminus of the therapeutic protein, or at a place between thetermini, if it is determined that such placement would not destroy therequired biological function of either the therapeutic protein or theKDR or VEGF/KDR complex binding polypeptide. A particular advantage ofthis general approach is that concatamerization of multiple, tandemlyarranged KDR or VEGF/KDR complex binding polypeptides is possible,thereby increasing the number and concentration of KDR or VEGF/KDRcomplex binding sites associated with each therapeutic protein. In thismanner KDR or VEGF/KDR complex binding avidity is increased, which wouldbe expected to improve the efficacy of the recombinant therapeuticfusion protein.

Similar recombinant proteins containing one or more coding sequences fora KDR and VEGF/KDR complex binding polypeptide may be useful in imagingor therapeutic applications. For example, in a variation of thepre-targeting applications discussed infra, the coding sequence for aKDR or VEGF/KDR complex binding peptide can be fused in frame to asequence encoding an antibody (or an antibody fragment or recombinantDNA construct including an antibody, etc.) that, for example, binds to achelator for a radionuclide (or another detectable label). The antibodyexpressing the KDR or VEGF/KDR complex binding polypeptide is thenadministered to a patient and allowed to localize and bind toKDR-expressing tissue. After the non-binding antibodies have beenallowed to clear, the chelator-radionuclide complex (or other detectablelabel), which the antibody recognizes is administered, permittingimaging of or radiotherapy to the KDR-expressing tissues.

Additionally, the coding sequence for a KDR or VEGF/KDR complex bindingpeptide may be fused in frame to a sequence encoding, for example, serumproteins or other proteins that produce biological effects (such asapoptosis, coagulation, internalization, differentiation, cellularstasis, immune system stimulation or suppression, or combinationsthereof). The resulting recombinant proteins are useful in imaging,radiotherapy, and therapies directed against cancer and other diseasesthat involve angiogenesis or diseases associated with the pathogensdiscussed herein.

Additionally, constructs including KDR or KDR/VEGF complex bindingpolypeptides of the present invention can themselves be used astherapeutics to treat a number of diseases. For example, where bindingof a protein or other molecule (e.g., a growth factor, hormone etc.) isnecessary for or contributes to a disease process and a binding moietyinhibits such binding, constructs including such binding moieties couldbe useful as therapeutics. Similarly, where binding of a binding moietyitself inhibits a disease process, constructs containing such bindingmoieties could also be useful as therapeutics.

As binding of VEGF and activation of KDR is necessary for angiogenicactivity, in one embodiment constructs including KDR complex bindingpolypeptides that inhibit the binding of VEGF to KDR (or otherwiseinhibit activation of KDR) may be used as anti-angiogenic agents. Somepeptides of the invention that inhibit activation of KDR are discussedin Example 9 infra. Certain constructs of the invention includingmultimers and heteromultimers that inhibit activation of KDR are alsodiscussed in the Examples. A particularly preferred heteromultimer isthe heterodimer-containing construct D1 (structures provided by theexamples). Other preferred heterodimer constructs include D4, D5, D6,D10, D13, D17, D23, D27, D30 and D31 (structures provided in theExamples below). The binding polypeptides and constructs thereof of thepresent invention are useful as therapeutic agents for treatingconditions that involve endothelial cells. Because an important functionof endothelial cells is angiogenesis, or the formation of blood vessels,the polypeptides and constructs thereof are particularly useful fortreating conditions that involve angiogenesis. Conditions that involveangiogenesis include, for example, solid tumors, tumor metastases andbenign tumors. Such tumors and related disorders are well known in theart and include, for example, melanoma, central nervous system tumors,neuroendocrine tumors, sarcoma, multiple myeloma as wells as cancer ofthe breast, lung, prostate, colon, head & neck, and ovaries. Additionaltumors and related disorders are listed in Table I of U.S. Pat. No.6,025,331, issued Feb. 15, 2000 to Moses, et al., the teachings of whichare incorporated herein by reference. Benign tumors include, forexample, hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas. As shown in Example 15, compounds of the invention,including heteromultimers such as D6, are useful in treating and/orslowing the growth of certain tumors.

Other relevant diseases that involve angiogenesis include for example,rheumatoid arthritis, psoriasis, and ocular diseases, such as diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rebeosis, Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibroma andwound granulation. Other relevant diseases or conditions that involveblood vessel growth include intestinal adhesions, atherosclerosis,scleroderma, and hypertropic scars, and ulcers. Furthermore, the bindingpolypeptides and constructs thereof of the present invention can be usedto reduce or prevent uterine neovascularization required for embryoimplantation, for example, as a birth control agent. Heteromultimers ofthis invention can also be useful for treating vascular permeabilityevents that can result when VEGF binds KDR. In renal failure, forexample, it has been shown that anti-VEGF antibodies can reverse damage.In a similar way, the compounds of the present invention can reverserenal permeability pathogenesis in, for example, diabetes.

Furthermore, the KDR or VEGF/KDR complex binding polypeptides of thepresent invention may be useful in treating diseases associated withcertain pathogens, including, for example, malaria, HIV, SIV, Simianhemorrhagic fever virus, etc. Sequence homology searches of KDR-bindingpeptides identified by phage display using the BLAST program at NCBI hasidentified a number of homologous proteins known or expected to bepresent on the surface of pathogenic organisms. Homologies were notedbetween the polypeptides of the invention and proteins from variousmalaria strains, HIV, SIV, simian hemorrhagic fever virus, and anenterohemorrhagic E. coli strain. Some of the homologous proteins, suchas PfEMP1 and EBL-1, are hypermutable adhesion proteins known to playroles in virulence. These proteins possess multiple binding sites thatare capable of binding to more than one target molecule on the host'ssurface. Their high mutation and recombination rates allow them toquickly develop new binding sites to promote survival and/or invasion.Similarly, proteins such as gp120 of HIV (which also has homology tosome of the KDR-binding peptides disclosed herein) play critical rolesin the adhesion of pathogens to their hosts. Although not reportedpreviously, it is possible that many of the pathogen proteins withhomology to the KDR-binding peptides disclosed herein also bind to KDR.Comparison of the pathogen protein sequences with the correspondingpeptide sequences may suggest changes in the peptide sequence or othermodifications that will enhance its binding properties. Additionally,the KDR-binding peptide sequences disclosed herein may have usefulnessin blocking infection with the pathogen species that possesses thehomology. Indeed, a similar strategy is being employed to block HIVinfection by trying to prevent virus envelope proteins from binding totheir known cellular surface targets such as CD4. See, Howie et al.,“Synthetic peptides representing discontinuous CD4 binding epitopes ofHIV-1 gp120 that induce T cell apoptosis and block cell death induced bygp120”, FASEB J, 12(11):991-998 (1998). Thus, KDR may represent apreviously unknown target for a number of pathogens, and the KDR bindingpeptides of the invention may be useful in treating the diseasesassociated with those pathogens.

The binding polypeptides and constructs thereof can be administered toan individual over a suitable time course depending on the nature of thecondition and the desired outcome. The binding polypeptides andconstructs thereof can be administered prophylactically, e.g., beforethe condition is diagnosed or to an individual predisposed to acondition. The binding polypeptides and constructs thereof can beadministered while the individual exhibits symptoms of the condition orafter the symptoms have passed or otherwise been relieved (such as afterremoval of a tumor). In addition, the binding polypeptides andconstructs thereof of the present invention can be administered a partof a maintenance regimen, for example to prevent or lessen therecurrence or the symptoms or condition. As described below, the bindingpolypeptides and constructs thereof of the present invention can beadministered systemically or locally.

The quantity of material administered will depend on the seriousness ofthe condition. For example, for treatment of an angiogenic condition,e.g., in the case of neoplastic tumor growth, the position and size ofthe tumor will affect the quantity of material to be administered. Theprecise dose to be employed and mode of administration must per force inview of the nature of the complaint be decided according to thecircumstances by the physician supervising treatment. In general,dosages of the agent conjugate of the present invention will follow thedosages that are routine for the therapeutic agent alone, although theimproved affinity of a binding polypeptide or heteromultimer of theinvention for its target may allow a decrease in the standard dosage.

Such conjugate pharmaceutical compositions are preferably formulated forparenteral administration, and most preferably for intravenous orintra-arterial administration. Generally, and particularly whenadministration is intravenous or intra-arterial, pharmaceuticalcompositions may be given as a bolus, as two or more doses separated intime, or as a constant or non-linear flow infusion.

As used herein the term “therapeutic” includes at least partialalleviation of symptoms of a given condition. The binding polypeptidesand constructs thereof of the present invention do not have to produce acomplete alleviation of symptoms to be useful. For example, treatment ofan individual can result in a decrease in the size of a tumor ordiseased area, or prevention of an increase in size of the tumor ordiseased area. Treatment can result in reduction in the number of bloodvessels in an area of interest or can prevent an increase in the numberof blood vessels in an area of interest. Treatment can also prevent orlessen the number or size of metastatic outgrowths of the main tumor(s).

Symptoms that can be alleviated include physiological characteristicssuch as VEGF receptor activity and migration ability of endothelialcells. The binding polypeptides and constructs thereof of the presentinvention can inhibit activity of VEGF receptors, including VEGFR-2/KDR,VEGFR-1/Flt-1 and VEGFR-3/Flt-4. Such inhibition can be detected, forexample, by measuring the phosphorylation state of the receptor in thepresence of or after treatment with the binding polypeptides orconstructs thereof. Such inhibition can also be detected by measuringthe ability of endothelial cells to migrate in the presence of or aftertreatment with the binding polypeptides or constructs thereof. Based onthe teachings provided herein, one of ordinary skill in the art wouldknow how and be able to administer a suitable dose of bindingpolypeptide or construct thereof as provided herein, and measure theeffect of treatment on the parameter of interest. For example, the sizeof the area of interest (e.g., the tumor or lesion) can be measuredbefore and after treatment. In another embodiment, the phosphorylationstate of the relevant receptor, or the migration ability of endothelialin an area of interest can be measured in samples taken from theindividual. The VEGF receptors or endothelial cells can be isolated fromthe sample and used in assays described herein.

The dosage of the polypeptides and constructs thereof may depend on theage, sex, health, and weight of the individual, as well as the nature ofthe condition and overall treatment regimen. The biological effects ofthe polypeptides and constructs thereof are described herein. Therefore,based on the biological effects of the binding polypeptides andconstructs provided herein, and the desired outcome of treatment, thepreferred dosage is determinable by one of ordinary skill in the artthrough routine optimization procedures. Typically, the daily regimen isin the range of about 0.1 μg/kg to about 1 mg/kg.

The binding polypeptides and constructs thereof provided herein can beadministered as the sole active ingredient together with apharmaceutically acceptable excipient, or can be administered togetherwith other binding polypeptides and constructs thereof, othertherapeutic agents, or combination thereof. In addition, the bindingpolypeptides and constructs thereof can be conjugated to therapeuticagents, for example, to improve specificity, residence time in the body,or therapeutic effect. Such other therapeutic agents include, forexample, other anti-angiogenic compounds, and tumoricidal compounds. Thetherapeutic agent can also include antibodies.

Furthermore, the binding polypeptide or constructs thereof of thepresent invention can be used as an endothelial cell homing device.Therefore, the binding polypeptide or constructs thereof can beconjugated to nucleic acid encoding, for example, a therapeuticpolypeptide, in order to target the nucleic acid to endothelial cells.Once exposed to the nucleic acid conjugated binding polypeptide, theendothelial cell can internalize and express the conjugated nucleicacid, thereby delivering the therapeutic peptide to the target cells.

In another embodiment of the invention, the therapeutic agent can beassociated with an ultrasound contrast agent composition, saidultrasound contrast agent including the KDR or VEGF/KDR complex bindingpeptides of the invention linked to the material employed to form thevesicles (particularly microbubbles or microballoons) comprising thecontrast agent. For example, the therapeutic agent can be associatedwith the contrast agent and delivered as described in U.S. Pat. No.6,258,378, herein incorporated by reference. Thus, after administrationof the ultrasound contrast agent and the optional imaging of thecontrast agent bound to the pathogenic site expressing the KDR orVEGF/KDR complex, the pathogenic site can be irradiated with an energybeam (preferably ultrasonic, e.g., with a frequency of from 0.3 to 3MHz), to rupture or burst of microvesicles. The therapeutic effect ofthe therapeutic agent can thus be advantageously enhanced by the energyreleased by the rupture of the microvesicles, in particular causing aneffective deliver of the therapeutic agent to the targeted pathogenicsite.

The binding polypeptides and constructs thereof can be administered byany suitable route. Suitable routes of administration include, but arenot limited to, topical application, transdermal, parenteral,gastrointestinal, intravaginal, and transalveolar. Compositions for thedesired route of administration can be prepared by any of the methodswell known in the pharmaceutical arts, for example, as described inRemington: The Science and Practice of Pharmacy, 20^(th) ed.,Lippincott, Williams and Wilkins, 2000.

For topical application, the binding polypeptides can be suspended, forexample, in a cream, gel or rinse that allows the polypeptides orconstructs to penetrate the skin and enter the blood stream, forsystemic delivery, or contact the area of interest, for localizeddelivery. Compositions suitable for topical application include anypharmaceutically acceptable base in which the polypeptides are at leastminimally soluble.

For transdermal administration, the polypeptides can be applied inpharmaceutically acceptable suspension together with a suitabletransdermal device or “patch.” Examples of suitable transdermal devicesfor administration of the polypeptides of the present invention aredescribed, for example, in U.S. Pat. No. 6,165,458, issued Dec. 26, 2000to Foldvari, et al., and U.S. Pat. No. 6,274,166B1, issued Aug. 4, 2001to Sintov, et al., the teachings of which are incorporated herein byreference.

For parenteral administration, the polypeptides can be injectedintravenously, intramuscularly, intraperitoneally, or subcutaneously.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Other pharmaceutically acceptablecarriers include, but are not limited to, sterile water, salinesolution, and buffered saline (including buffers like phosphate oracetate), alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, paraffin, etc.Where necessary, the composition may also include a solubilizing agentand a local anaesthetic such as lidocaine to ease pain at the site ofthe injection, preservatives, stabilizers, wetting agents, emulsifiers,salts, lubricants, etc. as long as they do not react deleteriously withthe active compounds. Similarly, the composition may compriseconventional excipients, i.e. pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral orintranasal application that do not deleteriously react with the activecompounds. Generally, the ingredients will be supplied either separatelyor mixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent inactivity units. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade “water for injection” or saline. Where thecomposition is to be administered by injection, an ampoule of sterilewater for injection or saline may be provided so that the ingredientsmay be mixed prior to administration.

For gastrointestinal and intravaginal administration, the polypeptidescan be incorporated into pharmaceutically acceptable powders, pills orliquids for ingestion, and suppositories for rectal or vaginaladministration.

For transalveolar, buccal or pulmonary administration, the polypeptidescan be suspended in a pharmaceutically acceptable excipient suitable foraerosolization and inhalation or as a mouthwash. Devices suitable fortransalveolar administration such as atomizers and vaporizers are alsoincluded within the scope of the invention. Suitable formulations foraerosol delivery of polypeptides using buccal or pulmonary routes can befound, for example in U.S. Pat. No. 6,312,665B1, issued Nov. 6, 2001 toPankaj Modi, the teachings of which are incorporated herein byreference.

In addition, the polypeptides of the present invention can beadministered nasally or ocularly, where the polypeptide is suspended ina liquid pharmaceutically acceptable agent suitable for dropwise dosing.

The polypeptides of the present invention can be administered such thatthe polypeptide is released in the individual over an extended period oftime (sustained or controlled release). For example, the polypeptide canbe formulated into a composition such that a single administrationprovides delivery of the polypeptide for at least one week, or over theperiod of a year or more. Controlled release systems include monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelles, liposomes, transdermal patches and iontophoreticdevices. In one embodiment, the polypeptides of the present inventionare encapsulated or admixed in a slowly degrading, non-toxic polymer.Additional formulations suitable for controlled release of thepolypeptides provided herein are described in U.S. Pat. No. 4,391,797,issued Jul. 5, 1983, to Folkman, et al., the teachings of which areincorporated herein by reference.

Another suitable method for delivering the polypeptides of the presentto an individual is via in vivo production of the polypeptide. A geneencoding the polypeptide can be administered to the individual such thatthe encoded polypeptide is expressed. The gene can be transientlyexpressed. In a particular embodiment, the gene encoding the polypeptideis transfected into cells that have been obtained from the patient, amethod referred to as ex vivo gene therapy. Cells expressing thepolypeptide are then returned to the patient's body. Methods of ex vivogene therapy are well known in the art and are described, for example,in U.S. Pat. No. 4,391,797, issued Mar. 21, 1998 to Anderson, et al.,the teachings of which are incorporated herein by reference.

Isolation, formulation and use of KDR or VEGF/KDR complex bindingmoieties in accordance with this invention will be further illustratedin the following Examples to follow.

As previously mentioned, the present invention also provides monomericpeptide phospholipid conjugates having a linear peptide monomer whichbinds with high affinity to KDR and dimeric peptide phospholipidconjugates having two distinct monomer subunits, each binding to KDR. Inaddition, highly efficient methods for large scale production ofpurified forms of these conjugates and precursor materials are provided.Such methods include the production of dimeric peptide phospholipidconjugates having minimal levels of TFA.

The phospholipid may be selected from the group consisting of:phosphatidylethanolamines and modified phosphatidylethanolamines.Particularly preferred phospholipids include phosphatidylethanolaminesmodified by linking a hydrophilic polymer thereto. Examples of modifiedphosphatidylethanolamines are phosphatidylethanolamines (PE) modifiedwith polyethylenglycol (PEG), in brief “PE-PEGs”, i.e.phosphatidylethanolamines where the hydrophilic ethanolamine moiety islinked to a PEG molecule of variable molecular weight (e.g. from 300 to5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG.DSPE-PEG2000, DSPE-PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are preferred,with DSPE-PEG2000 particularly preferred. Note that a salt form of thephospholipid may be used, such as, for example, the trimethyl ammoniumsalt, the tetramethylammonium salt, the triethylammonium salt, sodiumsalt, etc.

These compounds may be incorporated into gas-filled ultrasound contrastagents, such as, for example, gas filled microbubbles to form contrastagents that provide excellent imaging of target-bearing tissue. In apreferred embodiment, targeting vector-phospholipid conjugates whichinclude targeting peptides which bind with high affinity to KDR areincorporated into targeted microbubbles. As shown herein, such targetedmicrobubbles selectively localize at KDR-bearing tissue, permittingimaging of such tissue, and, in particular imaging of tumors andangiogenic processes, including those processes associated withneoplastic development.

Monomer Conjugates

Generally

Table 1-A provides a description for the identification labels shown inFIGS. 105, 106, 113 and 114.

TABLE 1-A 1Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH₂ (SEQ ID NO. 618)2 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ ID NO. 619) 3mono-NHS ester of glutaryl-peptide monomer (2)Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(NHS-Glut)-NH₂ (SEQ ID NO. 620) 4DSPE-PEG2000-NH₂ phospholipid1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-amine31Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH₂ (SEQ ID NO. 621)32 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ ID NO. 622)

As shown if FIGS. 105 and 106 the monomeric peptide phospholipidconjugate (1)N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-Ll-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysinamide,is a phospholipid conjugate. This conjugate is also referred to asAc-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO.618) andAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2.It comprises a 29 amino acid linear peptide monomer (2)N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L1-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide,also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO.619) andAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2.This novel peptide monomer binds with high affinity to KDR. It should beunderstood that analogs and derivatives of the monomeric peptidephospholipid conjugate (1) and the linear peptide monomer (2) areintended to be included within the scope of the present invention.

FIG. 114 provides the structure of another monmeric peptide phospholipidconjugate (31),N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysine-amide,a phospholipid conjugate. This conjugate is also referred to asAc-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO.621) andAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2.As shown in FIG. 113, the conjugate comprises a 28 amino acid linearpeptide monomer (32),N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide,which is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ IDNO.622) andAc-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2.This peptide monomer has been shown to bind with high affinity to KDR(See U.S. application Ser. No. 10/661,156). It should be understood thatanalogs and derivatives of the monomeric peptide phospholipid conjugateand the linear peptide monomer are intended to be included within thescope of the present invention.

Additional monomeric peptide phospholip conjugates according to thepresent invention include Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. IDNO. 623) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 624).These peptide monomers bind with high affinity to KDR. It should beunderstood that analogs and derivatives of these monomeric peptidemonomers and peptide phospholipids conjugates comprising these monomersare intended to be included within the scope of the present invention.

As shown in the Examples, ultrasound contrast agents such as gas filledmicrobubbles formulated with the monomeric peptide phospholipidconjugates (1) and (31) displayed high KDR binding which was confirmedusing echographic examination of VX2 tumors in rabbits.

Methods of Preparation of Monomer Peptide-Phospholipid Conjugates

Ideally, to facilitate production of a monomeric peptide phospholipidconjugate, the linear peptide monomer should be prepared in bulk. Thenconjugation of the purified linear peptide monomer to the phospholipid,such as, for example, a pegylated phospholipid in salt form, e.g.,DSPE-PEG2000-NH₂ phospholipid ammonium salt via the linkerdisuccinimidyl glutarate (DSG), may be used to provide monomeric peptidephospholipid conjugates.

In preparing monomeric peptide phospholipid conjugates, methodsaccording to the present invention provide at least the followingadvantages: increased yield of peptide synthesis; reduced extent ofracemization; avoidance of previously observed piperidine amideformation during synthesis, efficient purification of peptide monomers,development of a procedure for conjugation of peptide monomers on largerscale; and development of purification protocols that would allow theready separation of the monomeric peptide phospholipid conjugates fromthe starting DSPE-PEG2000-NH₂ phospholipid ammonium salt.

With respect to the methods described below (including the Examplessection of this specification), it should be understood that while suchmethods may specifically reference the use of linear peptide monomers(2) and (32) to form monomeric peptide phospholipids conjugates (1) and(31), these methods also may be used with peptide monomersAc-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 623) andAc-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 624), as well asanalogs and derivatives of any of the above. It also should beunderstood that the numerical values referred to in the synthesis ofmonomeric peptide phospholipid conjugates are representative.

Linear peptide monomers may be prepared by SPPS. The sequence of thelinear peptide monomers may be constructed as a C-terminal carboxamideon Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Peptide synthesismay be accomplished using Fmoc chemistry on a SONATA®/Pilot PeptideSynthesizer. Problems previously observed with this process have beenracemization, incomplete couplings and piperidine amide formation, eachof which contribute to suboptimal yield and purity. A dramatic decreasein the formation of the piperidine amide may be attained by the use of25% piperidine in DMF containing HOBt (0.1M) as the reagent for Fmocremoval. Racemization may be considerably reduced by using DIC/HOBt asthe activator for most couplings; a 3 h coupling time using a four-foldexcess of pre-activated Fmoc-amino acid with an intervening wash withanhydrous DMF (6×). N^(α)-Fmoc amino acids may be dissolved just beforetheir coupling turn and pre-activated with DIC/HOBt in DMF for 4 min andtransferred to the reaction vessel. This may be accomplished on theSonata instrument by loading the solid Fmoc-amino acids into the aminoacid vessels of the instrument and then programming the instrument toadd DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of thesolution.

To optimize the yield, the problem of aggregation of the resin duringthe synthesis of longer peptides, which can be devastating even whenoptimal coupling reagents are employed, may be addressed. To reduceaggregation during peptide assembly the strategy of using pseudoprolinedipeptides to incorporate X-Thr or X-Ser as dipeptides instead ofsequential couplings of X and Thr or X and Ser, may be employed. Forlinear peptide monomers sequential couplings of Leu11-Ser12 andLeu22-Ser23 may be replaced by the single coupling of the pseudoprolinedipeptide, Fmoc-Leu-Ser(ψMe,Mepro)-OH. Additional optimization may beaccomplished by reducing the number of couplings by usingFmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling ofFmoc-Gly-OH. Activation of -Gly-Gly-OH segments may lead to cyclizationof the activated acid function with the distal amide function to producean inactive diketopiperazine; this may reduce coupling yields in a timedependant manner. This problem may be avoided by addition ofFmoc-Glyn-OH (n=2, 3) to the reaction vessel and sequential addition ofHOBt and DIC; the activated Fmoc-Glyn-OH may be intercepted by theresin-bound amino group before appreciable cyclization to thediketopiperazine takes place. With these improvements, the synthesis oflinear peptide monomers may be completed on the Sonata PeptideSynthesizer on a 10 mmol synthesis scale.

After chain elongation, the Fmoc may be removed from the N-terminus. Thepeptide and the free amino group may be acetylated. Then the peptidesequence may be cleaved from the resin and deprotected using “Reagent B”(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. Afterthe cleavage reaction the crude peptide may be isolated as a solid byevaporation of the volatiles, trituration of the residue with diethylether and washing of the solid thus obtained using the same solvent. Inanother variation the peptide may be precipitated from the reactionmixture by addition of diethyl ether to the reaction mixture, collectingthe solid thus formed and washing with the same solvent.

Linear peptide monomers may be purified as described below. Again, thenumerical references are representative. Crude linear peptide monomers(0.5 g) may be dissolved in CH3CN (40 mL/g) and this solution may bediluted to a final volume of 100 mL with water. The solution may then befiltered. The filtered solution may be loaded onto the preparative HPLCcolumn (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm) equilibratedwith 10% CH3CN in water (0.1% TFA). After loading, the composition ofthe eluent may then be ramped to 20% CH3CN-water (0.1% TFA) over 1 min,and a linear gradient may be initiated at a rate of 0.6%/min of CH3CN(0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractionsmay be checked for purity on an analytical reversed phase C18 column(Waters XTerra MS-C18, 10, 120 Å, 4.6×50 mm) and fractions containingthe product in >95% purity may be combined and freeze-dried. For eachpurification of 0.5 g of crude peptide 0.12 g (24%) of linear peptidemonomer may be consistently isolated and will provide the peptide in thesame yield and purity.

Synthesis of monomeric peptide phospholipid conjugates may be performedas described below. The numerical references are again representative.The last step in the synthesis may be the conjugation of thephospholipid, such as, for example, a pegylated phospholipid such asDSPE-PEG2000-NH₂ phospholipid ammonium salt to a linear peptide monomer.The PEG2000 moiety of DSPE-PEG2000-NH₂ phospholipid ammonium salt (4) isnominally comprised of 45 ethylene glycol units. It should beunderstood, however, that this material is a distribution of PEGcontaining species whose centroid is the nominal compound containing 45ethylenoxy units. The conjugation of a linear peptide monomer withDSPE-PEG2000-NH2 phospholipid ammonium salt may be accomplished bypreparation of the glutaric acid monoamide mono NHS ester of a linearpeptide monomer and reaction of this with the free amino group of thephospholipid ammonium salt. Thus a linear peptide monomer may be reactedwith DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. Thereaction mixture may be diluted with ethyl acetate, which may result inprecipitation of the peptide glutaric acid monoamide mono-NHS ester. Thesupernatant containing un-reacted DSG may be decanted and theintermediate peptide mono-NHS ester may be washed several times withethyl acetate to remove traces of DSG. Mass spectral data confirms theformation of the peptide mono-NHS ester as a clean product. The solidmono-NHS ester may be dissolved in DMF and reacted with DSPE-PEG2000-NH₂phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for24 h. The linear peptide monomer glutaric acid monoamide mono-NHS estermay be used in excess to maximize the consumption of the phospholipidammonium salt because free phospholipid ammonium salt may complicate theisolation of monomeric peptide phospholipid conjugates in highly pureform.

The reaction mixture may be diluted with a 1:1 mixture of water (0.1%TFA) and CH₃CN—CH₃OH (1:1, v/v) (0.1% TFA) (˜100 mL), applied to areversed phase C2 column (Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm, flowrate 100 mL/min) and the column may be eluted with a 3:1 mixture ofwater (0.1% TFA) and CH₃CN—CH₃OH (1:1, v/v) (0.1% TFA) to removehydrophilic impurities. Then the product may be eluted using a gradientof CH₃CN—CH₃OH (1:1) (0.1% TFA) into water (0.1% TFA) (see ExperimentalSection for details). The collected fractions may be analyzed byreversed phase HPLC using an ELS detector which allows the detection ofthe desired product and the often difficult-to-separate DSPE-PEG2000-NH₂phospholipid which has very little UV absorbance. This indicates theclear separation of the monomeric peptide phospholipid conjugates andDSPE-PEG2000-NH₂ phospholipid. The pure product-containing fractions maybe collected, concentrated on a rotary evaporator (to reduce the contentof methanol) and freeze-dried to provide monomeric peptide phospholipidconjugates as a colorless solid. In order to prepare the requiredquantity of the monomeric peptide phospholipid conjugates, several runsmay be conducted employing 0.5 g to 1.0 g of linear peptide monomer. Inall cases the target monomeric peptide phospholipid conjugates may bewere isolated in high yield and purity (e.g., 57-60% yield and >99%purity).

Dimer Conjugate

Generally

Table 2-A provides a description for the identification labels shown inFIGS. 107, 108 and 109.

TABLE 2-A 11 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH₂ cyclic (2-12)disulfide}-NH₂ cyclic (6-13) disulfide 12Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ cyclic (6-13) disulfide 13Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide 14mono-NHS ester of glutaryl-peptide 12Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH₂ cyclic (6-13) disulfide15 ivDde-bearing dimerAc-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(ivDde)]-NH₂ cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide16 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH₂ cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide17 Mono-NHS ester of glutaryl-peptide 16Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(NHS-Glut)]-NH₂ cyclic (2-12) disulfide}-NH₂ cyclic (6-13)disulfide 18 DSPE-PEG2000-NH₂ phospholipid

As shown in those figures the dimeric peptide phospholipid conjugate(11)Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amidecyclic (2-12) disulfide]-amide cyclic (6-13) disulfide, consists of twomonomeric peptide chains which bind KDR: a 21 amino acid cyclicdisulfide peptide monomer (13)Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl)amidecyclic (2-12) disulfide, and a 22 amino acid cyclic disulfide peptidemonomer (12)Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysinamidecyclic 6-13 disulfide tethered by a glutaryl linker. It should beunderstood that analogs and derivatives of the dimeric peptidephospholipid conjugate (11) and the cyclic disulfide peptide monomers(12) and (13) are intended to be included within the scope of thepresent invention.

Ultrasound contrast agents (e.g. gas filled microbubbles) formulatedwith the dimeric peptide phospholipid conjugate (11) displayed high KDRbinding which was confirmed using echographic examination of VX2 tumorsin rabbits.

Methods of Preparation of Dimer-Phospholipid Conjugates

To accomplish synthesis of the dimeric peptide phospholipid conjugate(11), the monomers used for this purpose optimally should be prepared inbulk. Then the monomers may be tethered to each other usingdi-succinimidyl glutarate as a linker to form the precursor dimerpeptide (16),Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amidecyclic (2-12) disulfide]-amide cyclic (6-13) disulfide. Then conjugationof the purified precursor dimer peptide (16) to a DSPE-PEG2000-NH₂phospholipid ammonium salt (18) again via disuccinimidyl glutarate maybe used in order to provide the target dimeric peptide phospholipidconjugate (11).

In preparing dimeric peptide phospholipid conjugate (11), methodsaccording to the present invention provide at least the followingadvantages: increased yield of automated chain elongation of the peptidesequences; reduced extent of racemization encountered during synthesis;avoidance of previously observed piperidine amide formation duringsynthesis of peptide monomer (13); cyclization of linear di-cysteinecontaining peptide precursors of (12) and (13) using procedures amenableto multigram scale yet allowing efficient and practical sample handling;efficient purification of monomer peptides (12) and (13); maximizedyield and purity of precursor dimer peptide (16); development of aprocedure for conjugation of the precursor dimer peptide (16) on largerscale; and development of purification protocols that would allow theready separation of the target dimeric peptide phospholipid conjugate(11) from phospholipid ammonium salt (18).

The dimeric peptide phospholipid conjugate (11) may be prepared byautomated synthesis of the peptide monomers (12),Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2cyclic (6-13) disulfide, and (13),Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH₂cyclic (2-12) disulfide, their efficient coupling using disuccinimidylglutarate (DSG) to give an ivDde-protected dimer, its deprotection andsubsequent coupling to DSPE-PEG2000-NH₂, also via a glutaryl linkage.Using procedures according to the present invention, monomer peptidesmay be synthesized on a 10 mmol scale without complication and afterHPLC purification may be obtained in about 20% yield and >95% purity.Such methods allow dimer formation reactions and the subsequentconjugation to the phospholipid component providing formation of dimericpeptide phospholipid conjugate (11) to be carried out on a gram scale.The precursor dimer peptide (16) may be obtained from the monomerpeptides routinely in about 32% yield and >95% purity. The dimericpeptide phospholipid conjugate (11) may be produced from the precursordimer peptide (16) in 57-60% yield and >99% purity.

Dimeric peptide phospholipid conjugates may be prepared as describedbelow. It should be appreciated that the numerical values referred to inthis representative description of the synthesis of dimeric peptidephospholipid conjugates are representative.

Described below is a representative method for the solid phase synthesisand disulfide cyclization of a peptide monomer (12)Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2cyclic (6-13) disulfide, and a peptide monomer (13),Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2cyclic (2-12) disulfide.

The peptides may be constructed as their C-terminal carboxamides onPal-Peg-PS-resin (substitution level: 0.2 mmol/g). Chain elongation maybe accomplished using Fmoc chemistry employing optimized deprotectionand coupling protocols on a SONATA®/Pilot Peptide Synthesizer on a 10mmol synthesis scale. The optimized synthesis of the peptides byautomated SPSS may be developed by study of peptide impurities and theeffect of changes of particular elements of the protocols on the overallyield and purity of the peptides obtained.

Analysis of the impurities obtained from nonoptimized syntheses of themonomer peptides indicates that the major problems are racemization,incomplete couplings and piperidine amide formation (presumably via anintermediate aspartimide or glutarimide intermediate), each of whichcontributes to suboptimal yield and purity. A dramatic decrease information of the piperidine amide may be attained by the use of 25%piperidine in DMF containing HOBt (0.1M) as the reagent for fmocremoval. Racemization may be considerably reduced by using DIC/HOBt asthe activator for most couplings; and a 3 h coupling time using afour-fold excess of pre-activated Fmoc-amino acid with an interveningwash with anhydrous DMF (6×). N-^({tilde over (α)})Fmoc amino acids maybe dissolved just before their coupling turn and pre-activated withDIC/HOBt in DMF for 4 min and transferred to the reaction vessel. Thismay be accomplished on the Sonata instrument by loading the solidFmoc-amino acids into the amino acid vessels of the instrument and thenprogramming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentiallywith bubbling of the solution after each addition.

To optimize the yield, the problem of aggregation of the resin duringthe synthesis of longer peptides, which can be devastating even whenoptimal coupling reagents are employed, may be addressed. To reduceaggregation during peptide assembly the strategy of using pseudoprolinedipeptides to incorporate X-Thr or X-Ser (X refers to the n−1 amino acidof the sequence) as dipeptides instead of sequential couplings of X andThr or X and Ser, may be employed. Thus, for the monomer (12),Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2cyclic (6-13) disulfide, sequential coupling of suitably protected Thrand Gly (shown in bold above) may be replaced by the single coupling ofthe pseudoproline dipeptide, Fmoc-Gly-Thr(ψ^(Me,Me)pro)-OH. Similarly,in the synthesis of the monomer (13),Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2cyclic (2-12) disulfide, the pseudoproline dipeptide,Fmoc-Asp(OtBu)-Ser(ψ^(Me,Me)pro)-OH may be employed to replace thesequential coupling of suitably protected Ser and Asp (shown in boldfont above). Further optimization may be accomplished by reducing thenumber of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, inlieu of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OHsegments can lead to cyclization of the activated acid function with thedistal amide function to produce an inactive diketopiperazine; this mayreduce coupling yields in a time dependant manner. This problem may beavoided by addition of Fmoc-Glyn-OH (n=2, 3) to the reaction vessel andsequential addition of HOBt and DIC; the activated Fmoc-Glyn-OH may beintercepted by the resin-bound amino group before appreciablecyclization to the diketopiperazine takes place. After chain elongationis completed the N-terminal Fmoc protecting group may be removed fromeach of the peptides and the free amino group may be acetylated.

The pseudo-orthogonally protected derivative, Fmoc-Lys(ivDde)-OH may beused to enable the selective unmasking of the ε-amine of the C-terminallysine of the monomer and dimer peptides and their subsequentfunctionalization, which also may be optimized. The ivDde group on theε-amine of the C-terminal lysine of each of the peptide monomers may beremoved using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer (13) orLys(ivDde) for monomer (12) may be appended to the exposed lysineε-amino group using 4 equivalents of the Fmoc amino acid and 4equivalents each of DIC and HOBt in DMF for 10 h. After completion ofthe synthesis, the peptide sequence may be cleaved from the resin anddeprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane,88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction was complete thepeptide may be precipitated, washed with diethyl ether and dried.

The following procedures for cyclization of the linear di-cysteinecontaining peptides may be used to provide optimal scale-up of monomerpeptides. Generally the aerial oxidation of linear di-cysteine peptidesmay be carried out at ca 0.5-5 mg/mL (for the disclosed peptide monomers˜0.18-1.8 mM in peptide, ˜0.36-3.6 mM in cysteine thiol). In order towork at significantly higher concentrations DMSO-assisted cyclization ofdi-cysteine peptides allows the cyclization of ˜10 g of the linearpeptides in good yields in as little as ˜50 mL of solution. Thereforethe crude linear di-cysteine peptides may be cyclized in 95% DMSO-H2O (5mL/g) at pH 8.5 at ambient temperature. The progress of the cyclizationmay be routinely followed by mass spectroscopy and HPLC. Althoughcyclization may be essentially complete in ˜36 h, the reaction mixturemay be generally stirred for up to 48 h. The cyclic disulfide peptidesmay be precipitated from the reaction mixture by dilution with CH₃CN andthe resulting off-white crude solid peptides may be collected byfiltration. This is a convenient method for removing DMSO from the crudecyclic peptide.

Purification and isolation of monomer peptide (12),Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH₂ may be accomplished asdescribed below. Note that as used herein the designation “C*” refers toa cysteine residue that contributes to a disulfide bond. Attempts todissolve 0.5 g of the crude peptide in up to 300 mL of 30% CH₃CN inwater (0.1% TFA) have been unsuccessful. Therefore, as an alternative,the crude peptide, (0.5 g) may be dissolved in DMSO (5 mL/g) and thissolution may be diluted to a final volume of 100 mL with 20%CH₃CN-water. The solution may be filtered. The filtered solution may beloaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18,10μ, 300 Å, 50×250 mm) equilibrated with 10% CH₃CN (0.1% TFA) in water(0.1% TFA), and the column may be eluted with 10% CH₃CN (0.1% TFA) inwater (0.1% TFA) to wash DMSO from the column. The composition of theeluent then may be ramped to 35% CH₃CN-water (0.1% TFA) over 1 min, anda linear gradient may be initiated at a rate of 0.5%/min of CH₃CN (0.1%TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may bechecked for purity on an analytical reversed phase C18 column (WatersXTerra® MS-C18, 10μ, 120 Å, 4.6×50 mm) and fractions containing theproduct in >95% purity may be combined and freeze-dried. For eachpurification of 0.5 g of crude peptide 0.1 g (20%) for (12),Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH2 may be isolated. Repeatpurifications have been found to provide the peptide consistently in thesame yield and purity.

The peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂ maybe purified and isolated as described for peptide monomer (12) exceptthat the subject peptide may be dissolved in 20% CH₃CN (0.1% TFA) in0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a DMSO-containingdiluent. The resulting solution of crude peptide may be loaded onto thepreparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250mm, flow rate 100 mL/min) equilibrated with 10% CH₃CN in water (0.1%TFA). The column may be eluted with 10% CH₃CN (0.1% TFA)/water (0.1%TFA) at 100 mL/min for 5 min. Then the composition of the eluent may beramped to 30% CH₃CN (0.1% TFA)/water (0.1% TFA) over 1 min and a lineargradient rate of 0.5%/min of CH₃CN (0.1% TFA) into water (0.1% TFA) maybe initiated, and maintained until the desired peptide is completelyeluted from the column. Product-containing fractions may be analyzed ona Waters XTerra® analytical reversed phase C-18 column (10μ, 120 Å) andfractions containing the product in >95% purity may be pooled andfreeze-dried to afford the cyclic disulfide peptide monomer (13) (0.12g, 24% yield) in >95% purity. The 10 g of crude peptide monomer may bepurified serially in this manner.

Described below is a representative method for preparing the precursordimer peptide (16),Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[—NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide. The preparation ofthe precursor dimer peptide may be accomplished by the tethering of themonomer peptides in a two step procedure. First,Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH₂ (12) may be reacted withdisuccinimidyl glutarate (DSG, 5 eq.) in DMF in the presence of DIEA (5eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate,which results in precipitation of the glutaric acid monoamide mono-NHSester of the peptide. The supernatant, containing unreacted DSG, may bedecanted and the mono-NHS ester may be washed several times with ethylacetate to remove traces of DSG. Mass spectral data confirms theformation of the mono-NHS ester as a clean product. This may beredissolved in DMF and reacted with monomer peptideAc-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂ (13) in the presence of DIEA(5 eq). HPLC and MS results indicate the formation of the ivDde-bearingdimer, as a single major product. The ivDde protecting group on theε-amine of Lys of the dimer may be removed by stirring the reactionmixture with hydrazine (10%) in DMF for 20 min. The solution then may beacidified with TFA and diluted with 10% CH3CN (0.1% TFA)-water (0.1%TFA), applied to a preparative reversed phase C18 HPLC column andpurified by a gradient elution of acetonitrile (0.1% TFA) into 0.1%aqueous TFA. In order to provide the needed quantity of the precursordimer peptide, the reaction may be conducted employing from 0.5 g to asmuch as 1 g of each of the monomer peptides. In every case the requiredprecursor dimer peptide may be isolated in ˜32% yield and >95% purityconfirming the reproducibility and scalability of the procedures.

The final step in the synthesis may be the conjugation ofDSPE-PEG2000-NH₂ phospholipid ammonium salt (18) to the precursor dimerpeptide. As mentioned previously, the PEG2000 moiety of DSPE-PEG2000-NH2is nominally comprised of 45 ethylene glycol units. It should beunderstood, however, that this material is a distribution of PEGcontaining species whose centroid is the nominal compound containing 45ethylenoxy units.

Conjugation of the DSPE-PEG2000-NH₂ to the precursor dimer peptide maybe accomplished by preparation of a glutaric acid monoamide mono NHSester of the precursor dimer and reaction of this with the free aminogroup of the phospholipid ammonium salt. Thus the ivDde bearingprecursor dimer peptide (16) may be reacted with DSG (4 eq.) in DMF inthe presence of DIEA (5 eq.) for 30 min. As in the preparation of theprecursor dimer peptide the solution may be diluted with ethyl acetateto precipitate the glutaric acid monoamide mono-NHS ester of the dimer(17), as a solid. The supernatant may be decanted to remove theun-reacted DSG. The solid glutaric acid monoamide mono-NHS ester of thedimer peptide (17) may then be washed several times with ethyl acetateto remove traces of DSG. Mass spectral results confirm the formation ofthe glutaric acid monoamide mono-NHS ester of the peptide dimer as aclean product.

The dimer glutaric acid monoamide mono-NHS ester (17) may be dissolvedin DMF-CH2Cl2 (8:2) and reacted with DSPE-PEG2000-NH₂ phospholipidammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h. TheNHS ester (17) may be used in excess to maximize the consumption of thephospholipid ammonium salt because any free phospholipid may complicatethe purification and isolation of the final product. The reactionmixture may be diluted with water (0.1% TFA)-CH3CN—CH3OH (1:1) (0.1%TFA) (˜100 mL), applied to a reversed phase C4 column (Kromasil® PrepC4, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) and the column may beeluted with water (0.1% TFA)-CH₃CN—CH₃OH (1:1) (0.1% TFA) solventmixture to remove hydrophilic impurities. Then the product may be elutedusing a gradient of CH₃CN—CH₃OH (1:1) (0.1% TFA) into water (0.1% TFA).The collected fractions may be analyzed by reversed phase HPLC using anELS detector which allows the detection of the desired product and theoften difficult to separate DSPE-PEG2000-NH₂ phospholipid ammonium saltwhich has no strong UV chromophore. This indicates the clear separationof dimeric peptide phospholipid conjugate and DSPE-PEG2000-NH₂phospholipid ammonium salt. The pure product-containing fractions may becollected, concentrated on a rotary evaporator (to reduce the content ofmethanol) and freeze-dried to provide the dimer peptide phospholipidconjugate as a colorless solid.

In order to prepare the required quantity of the dimer peptidephospholipid conjugate, several runs may be conducted employing 0.5 g to1.0 g of the precursor dimer peptide. In all cases the target dimerpeptide phospholipid conjugate may be isolated in 57-60% yield andin >99% purity. The bulk quantity of dimer peptide phospholipidconjugate, obtained from the serial runs described above may be obtainedby dissolution of the product from the individual runs int-butanol-acetonitrile-water (1:1:3) followed by lyophilization. Theprocedure of Ellman for quantitative estimation of free thiol may beapplied to the bulk sample of the dimeric peptide phospholipidconjugate; free thiol, if present will be below the limit of detection.Amino acid composition analysis gives results within the acceptablelimits, supporting the assigned structure of the peptide derivative.MALDI-TOF mass spectral analysis also supports the presumed structure ofthe dimeric peptide phospholipid conjugate.

Methods of Preparation of Dimer-Phospholipid Conjugates Having Low orNegligible Levels of TFA

The present invention also provides methods for producing dimericpeptide-phospholipid conjugates having very low levels of TFA. Whilecertain methods provide for the synthesis and purification of suchconjugates on a gram scale, formation of a lyso-version of theconjugates has been observed upon storage of lyophilized material at 5°C. or upon storage of aqueous solutions of the conjugates. It isbelieved that the lyso-compound is formed by TFA-promoted acidhydrolysis of one of the phospholipid fatty acid esters in dimerpeptide-phospholipid conjugates.

To obtain the phospholipid peptide as a stable material bearing apharmaceutically acceptable counterion, highly efficient methods forobtaining dimer peptide-phospholipid conjugates were discovered whichconvert the TFA salts of the dimer peptide-phospholipid conjugate, orany suitable precursor(s), to analogous pharmaceutical acetate salt(s).Representative embodiments of these methods are provided below.

Table 3-A provides a description for the identification labels shown inFIGS. 110, 111 and 112.

TABLE 3-A 21 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH₂ cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide 22Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ cyclic (2-12) disulfide• nTFA 23Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ cyclic (2-12) disulfide• xHOAc24 mono-NHS ester of glutaryl-peptide 23Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH₂ cyclic (2-12) disulfide 25Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide• yTFA 26Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide• zHOAc27 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH₂ cyclic (2-12)disulfide]-NH₂ cyclic (6-13) disulfide•X HOAc 28Mono-NHS ester of glutaryl-peptide 27 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K(NHS-Glut)]-NH₂ cyclic(2-12) disulfide}-NH₂ cyclic (6-13) disulfide 29 DSPE-PEG2000-NH₂

Where m, n, x, y, z are variable depending on lyophilization conditions.

Referring now to FIGS. 110 and 111, in certain embodiments monomerpeptide components of heterodimer peptide (27), namely TFA saltscompounds (22) and (25), are subjected to ion exchange chromatography onthe macroporous sulfonic acid cation exchange resin AG MP-50 using astep gradient of ammonium acetate to convert them to their acetatesalts. Then the two peptide monomer acetates (23) and (26) may betethered through a glutaryl linker to form the dimer (27) as an acetatesalt. Purification of the crude dimer acetate salt of (27), by C-18preparative HPLC using a linear gradient method employing CH₃CN H₂O eachcontaining 10 mM NH4OAc provides the pure dimer acetate (27).Conjugation of this dimer to DSPE-PEG2000-NH₂ (29) and finalpurification of the crude mixture by C-3 preparative HPLC usingCH₃CN/H₂O/NH₄OAc provides compound (21) as the acetate salt.

More specifically, compounds (22), (25) and (27) all bear side-chaincarboxylic acid and amino groups. AG MP-50, a macroporouscation-exchange resin, may be used to allow full penetration of theresin by the peptides and to exploit the immobilization of the peptidesvia their basic (amino and guanidine groups). TFA salts of the peptidesmay be adsorbed to an AG MP-50 column (sulfonic acid form) and thecolumn may be washed with water and then eluted with a step gradient ofNH₄OAc in 0 or 30% CH₃CN/H₂O, depending on the solubility of thepeptides. The peptides may be eluted at about 600 mM NH₄OAc and theacetate form of the peptides then may be obtained in pure form. Both ICfluorine analysis and CE TFA counter-ion analysis consistently show verylow TFA content of the peptides.

Preferred methods also include redissolution/relyophilization of thefinal peptides several times to remove residual NH4OAc. Otherwise,residual traces of NH₄OAc present in the peptides may give rise to freeammonia in the presence of DIEA. This may result in the formation ofunwanted peptide-Glut-amide as a major product in subsequent preparationof (27) from the monomers (23) and (26) or final phospholipid-peptideconjugate (21) from the acetate salt of (27).

Referring now to FIG. 111, another embodiment provides the conversion ofthe TFA salt of dimer (27) to its analogous acetate salt by ion exchangechromatography on the macroporous sulfonic acid cation exchange resin AGMP-50. This dimer acetate then may be conjugated with DSPE-PEG2000-NH₂followed by purification of the crude material by C-3 preparative columnusing CH₃CN/H₂O/NH₄OAc to give the final compound (21) as an acetatesalt.

While the methods described above and in FIGS. 110 and 111 provideexcellent results, the second approach has the advantage of requiringfewer steps. Additional details are provided below in the Examplessection.

Turning to FIG. 112, another embodiment provides methods for providingdimeric conjugates having minimal amounts of TFA utilizing the sizedifferential between the phospholipid-peptide conjugate (21) and TFAions. In this embodiment 21●nTFA adduct may be eluted down a sizeexclusion column in the presence of ammonium bicarbonate buffer. Thecrude 21●nTFA initially may be freed of the lyso-compound by preparativeHPLC on a Zorbax C-3 column using a linear gradient of acetonitrile intowater. Both phases may be buffered with 10 mM ammonium acetate. Thisprovides separation of the lyso-compound as indicated by analyticalHPLC.

To further reduce the amount of TFA, the material may be applied to aSephadex G-25 column and eluted with aqueous ammonium bicarbonatesolution. The eluate may be monitored by HPLC. Product-containingfractions may be pooled and lyophilized to afford the desired material(21) essentially free of TFA and with high recovery rates. Additionaldetail is provided below in the Examples section.

Both the monomeric and dimeric peptide phospholipid conjugates describedherein may be incorporated into ultrasound contrast agents such as, forexample, gas filled microvesicles. Such gas filled microvesiclesinclude, for example, gas filled microbubbles, gas filled microballoons,gas filled microcapsules, etc. In a preferred embodiment, the peptidephospholipid conjugates may be incorporated into ultrasound contrastagents comprising gas filled microbubbles. Methods of preparation of gasfilled microbubbles from phospholipids and phospholipid conjugates areknown to those skilled in the art. For example, microbubbles accordingto the present invention can be prepared by methods described in any oneof the following patents: EP 554213, WO 04/069284, U.S. Pat. No.5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No.5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833,U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No.5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat.No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S.Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No.5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S.Pat. No. 5,908,610, which are incorporated by reference herein in theirentirety. The methods disclosed in WO 04/069284 are particularlypreferred.

Suitable phospholipids include esters of glycerol with one or twomolecules of fatty acids (the same or different) and phosphoric acid,wherein the phosphoric acid residue is in turn bonded to a hydrophilicgroup, such as choline, serine, inositol, glycerol, ethanolamine, andthe like groups. Fatty acids present in the phospholipids are in generallong chain aliphatic acids, typically containing from 12 to 24 carbonatoms, preferably from 14 to 22, that may be saturated or may containone or more unsaturations. Examples of suitable fatty acids are lauricacid, myristic acid, palmitic acid, stearic acid, arachidic acid,behenic acid, oleic acid, linoleic acid, and linolenic acid. Mono estersof phospholipids are known in the art as the “lyso” forms of thephospholipid.

Further examples of phospholipids are phosphatidic acids, i.e., thediesters of glycerol-phosphoric acid with fatty acids, sphingomyelins,i.e., those phosphatidylcholine analogs where the residue of glyceroldiester with fatty acids is replaced by a ceramide chain, cardiolipins,i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid,gangliosides, cerebrosides, etc.

As used herein, the term phospholipids includes either naturallyoccurring, semisynthetic or synthetically prepared products that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins. Examples of semisynthetic phospholipids are thepartially or fully hydrogenated derivatives of the naturally occurringlecithins.

Examples of synthetic phospholipids are e.g.,dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”),diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine(“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts,distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts,distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid(“DAPA”) and its alkali metal salts, dimyristoylphosphatidyl-ethanolamin-e (“DMPE”), dipalmitoylphosphatidylethanolamine (“DPPE”), distearoyl phosphatidyl-ethanolamine(“DSPE”), dimyristoyl phosphatidylserine (“DMPS”), diarachidoylphosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”),distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine(“DOPS”), dipalmitoyl sphingomyelin (“DPSP”), and distearoylsphingomyelin (“DSSP”).

Suitable phospholipids further include phospholipids modified by linkinga hydrophilic polymer thereto. Examples of modified phospholipids arephosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), inbrief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilicethanolamine moiety is linked to a PEG molecule of variable molecularweight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG,DMPE-PEG or DAPE-PEG (where DAPE is1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine). The compositionsalso may contain other amphiphilic compounds including, for instance,fatty acids, such as palmitic acid, stearic acid, arachidonic acid oroleic acid; sterols, such as cholesterol, or esters of sterols withfatty acids or with sugar acids; glycerol or glycerol esters includingglycerol tripalmitate, glycerol distearate, glycerol tristearate,glycerol dimyristate, glycerol trimyristate, glycerol dilaurate,glycerol trilaurate, glycerol dipalmitate; tertiary or quaternaryalkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammonium-propane(DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), andmixtures or combinations thereof.

Preferably, the formulation comprises at least one component bearing anoverall net charge, such as, for instance, phosphatidic acid, PE-PEG,palmitic acid, stearic acid, Ethyl-DSPC or DSTAP, preferably in a molaramount of less than about 50%. Particularly preferred formulations mayinclude mixtures of two or more of the following components: DSPC, DPPG,DPPA, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearic acid. Somepreferred phospholipids and formulations are set forth in the examples.Any of the gases disclosed herein or known to the skilled artisan may beemployed; however, inert gases, such as SF6 or perfluorocarbons likeCF₄, C₃F₈ and C₄F₁₀, are preferred, optionally in admixture with othergases such as air, nitrogen, oxygen or carbon dioxide

The preferred microbubble suspensions of the present invention may beprepared from phospholipids using known processes such as afreeze-drying or spray-drying solutions of the crude phospholipids in asuitable solvent or using the processes set forth in EP 554213; WO04/069284; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962;EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat.No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; U.S. Pat. No.5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat.No. 5,658,551; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,911,972; U.S.Pat. No. 6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No.5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat.No. 6,187,288; and U.S. Pat. No. 5,908,610, which are incorporated byreference herein in their entirety. Preferably, as disclosed inInternational patent application WO 04/069284, a microemulsion can beprepared which contains the phospholipids (e.g DSPC and/or DSPA) inadmixture with a lyoprotecting agent (such as, for instance,carbohydrates, sugar alcohols, polyglycols and mixtures thereof, asindicated in detail hereinafter) and optionally other amphiphilicmaterials (such as stearic acid), dispersed in an emulsion of water andof a water immiscible organic solvent. Preferred organic solvents arethose having solubility in water of 1.0 g/l or lower, preferably lowerabout 0.01 g/l, and include, for instance, pentane, hexane, heptane,octane, nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane,cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene,ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl etherand di-isopropylketone, chloroform, carbon tetrachloride,2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane,perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin,methylperfluorobutylether, methylperfluoroisobutylether,ethylperfluorobutylether, ethylperfluoroisobutylether and mixturesthereof. The peptide-phospholipid conjugate of the invention can beadmixed together with the phospholipid forming the microvesicle'senvelope, in the microemulsion. Preferably, an aqueous suspension of thepeptide-phospholipid conjugate and of a PE-PEG (e.g. DSPE-PEG2000) isfirst prepared, which is then admixed together with an aqueous-organicemulsion comprising the phospholipid and the lyoprotecting agent.Preferably said mixing is effected under heating, e.g. form about 40° C.to 80° C.

Prior to formation of the suspension of microbubbles by dispersion in anaqueous carrier, the freeze dried or spray dried phospholipid powdersare contacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments that will stabilize themicrobubbles of the gas dispersed therein. This method permitsproduction of suspensions of microbubbles that are stable even whenstored for prolonged periods and are obtained by simple dissolution ofthe dried laminarized phospholipids (which have been stored under adesired gas) without shaking or any violent agitation.

Alternatively, microbubbles can be prepared by suspending a gas into anaqueous solution at high agitation speed, as disclosed e.g. in WO97/29783. A further process for preparing microbubbles is disclosed inWO 2004/069284, herein incorporated by reference, which comprisespreparing an emulsion of an organic solvent in an aqueous medium in thepresence of a phospholipid and subsequently lyophilizing said emulsion,after optional washing and/or filtration steps. Some preferredpreparation methods are disclosed in the examples.

The formulation for the preparation of the gas-filled microbubbles mayadvantageously further comprise a lyophilization additive, such as anagent with cryoprotective and/or lyoprotective effect and/or a bulkingagent, for example an amino-acid such as glycine; a carbohydrate, e.g. asugar such as sucrose, mannitol, maltose, trehalose, glucose, lactose ora cyclodextrin, or a polysaccharide such as dextran; or a polyglycolsuch as polyethylene glycol (e.g. PEG-4000).

Any of these ultrasound compositions should also be, as far as possible,isotonic with blood. Hence, before injection, small amounts of isotonicagents may be added to any of above ultrasound contrast agentsuspensions. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2.6% glycerol solution, 5% dextrose solution, etc. Additionally, theultrasound compositions may include standard pharmaceutically acceptableadditives, including, for example, emulsifying agents, viscositymodifiers, cryoprotectants, lyoprotectants, bulking agents etc.

Any biocompatible gas may be used in the ultrasound contrast agents ofthe invention. The term “gas” as used herein includes any substances(including mixtures) substantially in gaseous form at the normal humanbody temperature. The gas may thus include, for example, air, nitrogen,oxygen, CO₂, argon, xenon or krypton, fluorinated gases (including forexample, perfluorocarbons, SF₆, SeF₆) a low molecular weight hydrocarbon(e.g., containing from 1 to 7 carbon atoms), for example, an alkane suchas methane, ethane, a propane, a butane or a pentane, a cycloalkane suchas cyclopropane, cyclobutane or cyclopentene, an alkene such asethylene, propene, propadiene or a butene, or an alkyne such asacetylene or propyne and/or mixtures thereof. However, fluorinated gasesare preferred. Fluorinated gases include materials that contain at leastone fluorine atom such as SF₆, freons (organic compounds containing oneor more carbon atoms and fluorine, i.e., CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀,CBrF₃, CCl₂F₂, C₂ClF₅, and CBrClF₂) and perfluorocarbons. The termperfluorocarbon refers to compounds containing only carbon and fluorineatoms and includes, in particular, saturated, unsaturated, and cyclicperfluorocarbons. The saturated perfluorocarbons, which are usuallypreferred, have the formula CnFn+2, where n is from 1 to 12, preferablyfrom 2 to 10, most preferably from 3 to 8 and even more preferably from3 to 6. Suitable perfluorocarbons include, for example, CF₄, C₂F₆,C₃F₈C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₂, C₇F₁₄, C₈F₁₈, and C₉F₂₀. Most preferablythe gas or gas mixture comprises SF₆ or a perfluorocarbon selected fromthe group consisting of C₃F₈ C₄F₈, C₄F₁₀, C₅F₂, C₆F₁₂, C₇F₁₄, C₈F₁₈,with C₄F₁₀ being particularly preferred. See also WO 97/29783, WO98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO98/18501, WO 98/05364, WO 98/17324. In a preferred embodiment the gascomprises C₄F₁₀ or SF₆, optionally in admixture with air, nitrogen,oxygen or carbon dioxide.

In certain circumstances it may be desirable to include a precursor to agaseous substance (e.g., a material that is capable of being convertedto a gas in vivo, often referred to as a “gas precursor”). Preferablythe gas precursor and the gas it produces are physiologicallyacceptable. The gas precursor may be pH-activated, photo-activated,temperature activated, etc. For example, certain perfluorocarbons may beused as temperature activated gas precursors. These perfluorocarbons,such as perfluoropentane, have a liquid/gas phase transition temperatureabove room temperature (or the temperature at which the agents areproduced and/or stored) but below body temperature; thus they undergo aphase shift and are converted to a gas within the human body.

As discussed above, the gas can comprise a mixture of gases. Thefollowing combinations are particularly preferred gas mixtures: amixture of gases (A) and (B) in which, at least one of the gases (B),present in an amount of between 0.5-41% by vol., has a molecular weightgreater than 80 daltons and is a fluorinated gas and (A) is selectedfrom the group consisting of air, oxygen, nitrogen, carbon dioxide andmixtures thereof, the balance of the mixture being gas A.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilized product may be stored andtransported without need of temperature control of its environment andin particular it may be supplied to hospitals and physicians for on siteformulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities. Preferably insuch a case it can be supplied in the form of a two-component kit, whichcan include two separate containers or a dual-chamber container. In theformer case preferably the container is a conventional septum-sealedvial, wherein the vial containing the lyophilized residue of step b) issealed with a septum through which the carrier liquid may be injectedusing an optionally prefilled syringe. In such a case the syringe usedas the container of the second component is also used then for injectingthe contrast agent. In the latter case, preferably the dual-chambercontainer is a dual-chamber syringe and once the lyophilizate has beenreconstituted and then suitably mixed or gently shaken, the containercan be used directly for injecting the contrast agent. In both casesmeans for directing or permitting application of sufficient bubbleforming energy into the contents of the container are provided.

However, as noted above, in the stabilised contrast agents according tothe invention the size of the gas microbubbles is substantiallyindependent of the amount of agitation energy applied to thereconstituted dried product. Accordingly, no more than gentle handshaking is generally required to give reproducible products withconsistent microbubble size.

It can be appreciated by one of ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g. a targeting ligand to belinked to the phospholipid during reconstitution), it can be packagedwith the other components of the kit, preferably in a form or containeradapted to facilitate ready combination with the other components of thekit.

No specific containers, vial or connection systems are required; thepresent invention may use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirable substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.The stopper may be a compound or multicomponent formulation based on anelastomer, such as poly(isobutylene) or butyl rubber.

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles can be administered, for example, in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.

Ultrasound imaging techniques that can be used in accordance with thepresent invention include known techniques, such as color Doppler, powerDoppler, Doppler amplitude, stimulated acoustic imaging, and two- orthree-dimensional imaging techniques. Imaging may be done in harmonic(resonant frequency) or fundamental modes, with the second harmonicpreferred.

The ultrasound contrast agents of the present invention may further beused in a variety of therapeutic imaging methods. The term therapeuticimaging includes within its meaning any method for the treatment of adisease in a patient which comprises the use of a contrast imaging agent(e.g. for the delivery of a therapeutic agent to a selected receptor ortissue), and which is capable of exerting or is responsible to exert abiological effect in vitro and/or in vivo. Therapeutic imaging mayadvantageously be associated with the controlled localized destructionof the gas-filled microvesicles, e.g. by means of an ultrasound burst athigh acoustic pressure (typically higher than the one generally employedin non-destructive diagnostic imaging methods). This controlleddestruction may be used, for instance, for the treatment of blood clots(a technique also known as sonothrombolysis), optionally in combinationwith the localized release of a suitable therapeutic agent.Alternatively, said therapeutic imaging may include the delivery of atherapeutic agent into cells, as a result of a transient membranepermeabilization at the cellular level induced by the localized burst ofthe microvesicles. This technique can be used, for instance, for aneffective delivery of genetic material into the cells; optionally, adrug can be locally delivered in combination with genetic material, thusallowing a combined pharmaceutical/genetic therapy of the patient (e.g.in case of tumor treatment).

The term “therapeutic agent” includes within its meaning any substance,composition or particle which may be used in any therapeuticapplication, such as in methods for the treatment of a disease in apatient, as well as any substance which is capable of exerting orresponsible to exert a biological effect in vitro and/or in vivo.Therapeutic agents thus include any compound or material capable ofbeing used in the treatment (including diagnosis, prevention,alleviation, pain relief or cure) of any pathological status in apatient (including malady, affliction, disease lesion or injury).Examples of therapeutic agents are drugs, pharmaceuticals, bioactiveagents, cytotoxic agents, chemotherapy agents, radiotherapeutic agents,proteins, natural or synthetic peptides, including oligopeptides andpolypeptides, vitamins, steroids and genetic material, includingnucleosides, nucleotides, oligonucleotides, polynucleotides andplasmids.

EXAMPLES Methods for the Examples

The following methods were employed in Examples 4-10. The followingcommon abbreviations are used: 9-fluorenylmethyloxycarbonyl (Fmoc),1-hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC),N-methylpyrrolidinone (NMP), acetic anhydride (Ac₂O),(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),trifluoroacetic acid (TFA), Reagent B (TFA:H₂O:phenol:triisopropylsilane 88:5:5:2), diisopropylethylamine (DIEA),O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), N-hydroxysuccinimide (NHS), solid phasepeptide synthesis (SPPS), dimethyl sulfoxide (DMSO), dichloromethane(DCM), dimethylformamide (DMF), human serum albumin (HSA), andradiochemical purity (RCP).

Method 1 for the ACT 357 MPS and ACT 496 MOS Synthesizers

The peptides were synthesized on NovaSyn TGR (Rink amide) resin (0.2mmol/g) using the Advanced ChemTech ACT 357 or ACT 496 Synthesizersemploying Fmoc peptide synthesis protocols, specifically using HOBt/DICas the coupling reagents and NMP as the solvent. The Fmoc was removed bytreating the Nova-Syn TGR (Rink amide-available from NovaBiochem, SanDiego Calif.) resin-bound peptide with 25% piperidine in DMF twice (4min and 10 min). All amino acids were dissolved in NMP (DMF was addedwhen the amino acid was not soluble in pure NMP). The concentration ofthe amino acid was 0.25M, and the concentrations for HOBt and DICrespectively were 0.5 M.

For a 0.04 mmol scale synthesis:

A typical amino acid coupling cycle (not including wash steps) was todispense piperidine solution (2.4 mL) to each well and mix for 4 min,then empty all wells. NMP (320 μL), HOBt solution (320 μL, 4 eq), aminoacid (640 μL, 4 eq) and DIC (320 μL, 4 eq) solutions were dispensed toeach well. The coupling time was 3 h; then the resin was washed. Thecycle was repeated for each amino acid. After the last amino acidcoupling, the resin-bound peptide was treated with 25% piperidine toremove the Fmoc protecting group. After washing, the resin bound peptidewas capped with 1.0M Ac₂O (1.2 mL per well) and diisopropylethylamine inDMF, optionally including varying amounts of HOBt in the mixture for 30min. The resin was washed with methanol and then dichloromethane anddried. Cleavage of the peptides from the resin and side-chaindeprotection was accomplished using Reagent B for 4.5 h. The cleavagesolutions were collected and the resins were washed with an additionalaliquot of Reagent B. The combined solutions were concentrated todryness. Ether was added to the residue with swirling or stirring toprecipitate the peptides. The ether was decanted, and solid wascollected. This procedure was repeated 2-3 times to remove impurities.The crude linear peptides were dissolved in DMSO and water mixtures, andpurified by HPLC (column: Waters Associates Xterra C18, 19×50 mm;solvents: H₂O with 0.1% TFA and CH₃CN with 0.1% TFA; UV 220 μm; Flowrate: 50-60 mL/min). The solutions containing the peptide werelyophilized to give the desired peptides as white fluffy lyophilizates(>90% purity). The purified linear di-cysteine containing peptides weredissolved in water, mixtures of water-acetonitrile, or mixtures ofwater-DMSO at concentrations between 0.1 mg/mL and 2.0 mg/mt. The choiceof solvent was a function of the solubility of the crude peptide in thesolvent. The pH of the solution was adjusted to pH 7.5-8.5 with aqueousammonia, aqueous ammonium carbonate or aqueous ammonium bicarbonate. Themixture was stirred vigorously in air for 24-48 hrs. In the case ofnon-DMSO containing solvent systems, the pH of the solution was adjustedto pH 2 with aqueous trifluoroacetic acid. The mixture was lyophilizedto provide the crude cyclic disulfide containing peptide. The cyclicdisulfide peptide was then dissolved to a volume of 1-2 mL in aqueous(0.1% TFA) containing a minimum of acetonitrile (0.1% TFA). Theresulting solution was loaded onto a reverse phase column and thedesired compound obtained by a gradient elution of acetonitrile intowater, employing a C18, or C8 reverse phase semipreparative orpreparative HPLC column. In the case of the DMSO-containing solutions,the solution was diluted until the DMSO concentration was minimalwithout precipitation of the peptide. The resulting mixture was quicklyacidified to pH 2 with dilute trifluoroacetic acid and loaded onto thereverse phase HPLC system and purified as described. Fractionscontaining the desired materials were pooled and the peptides isolatedby lyophilization.

Method 2 for the ACT 357 MPS and ACT 496 MOS Synthesizers

The peptides were synthesized as in Method 1 with the following changes.HBTU/HOBt/DIEA were used as the coupling reagent and NMP as the solvent.A low load (˜0.2 mmol/g) Fmoc-GGGK(Boc)-NovSyn-TGR-resin-prepared fromthe above-described Nova-Syn TGR resin was employed for peptidesynthesis on 0.01 mmol scale.

For a 0.01 mmol scale synthesis:

After the Fmoc group was removed, a standard coupling procedure used asolution of HOBt (720 μl, 6 eq), amino acid (804 μl, 6.6 eq), HBTU (720μl, 6 eq) and DIEA (798 μl, 13.3 eq). The mixture was agitated for 15min., emptied and the resin washed. After all couplings and aftercleavage and purification as above, the solutions containing desiredlinear peptides were lyophilized to give the peptides (>90% purity) aswhite fluffy solids. The crude ether-precipitated linear di-cysteinecontaining peptides were cyclized by dissolution in water, mixtures ofaqueous acetonitrile (0.1% TFA), or aqueous DMSO and adjustment of thepH of the solution to pH 7.5-8.5 by addition of aqueous ammonia, aqueousammonium carbonate, or aqueous ammonium bicarbonate solution. Thepeptide concentration was between 0.1 and 2.0 mg/mL. The mixture wasstirred in air for 24-48 hrs., acidified to a pH 2 with aqueoustrifluoroacetic acid, and then purified by preparative reverse phaseHPLC employing a gradient of acetonitrile into water. Fractionscontaining the desired material were pooled and the peptides wereisolated by lyophilization.

Method 3 for the ACT 496 MOS Synthesizer

The peptides were synthesized by using an Advanced ChemTech ACT 496 MOSSynthesizer as in method 1. The low load (˜0.2 mmol/g)GGGK(Boc)-NovaSyn-TGR resin was employed for peptide synthesis. Thecoupling solvent was NMP/DMSO 8:2. The synthesis was performed at a 0.02mmol scale using a coupling time of 3 h. The crude linear peptides werefurther processed as described for Method 1.

Method 4 for the ACT 496 MOS Synthesizer

The peptides were synthesized using method 3 on the ACT 496 withHBTU/DIEA as the coupling reagents, and NMP as the solvent.2,4,6-collidine as a 1 M solution was used as the base. The low loadFmoc-GGGK(ivDde)-Novsyn-TGR resin (˜0.2 mmol/g) was used for peptidesynthesis. The coupling time was 30 minutes. The crude linear peptideswere further processed as described for Method 1.

Method 5 for the ABI 433A Synthesizer

Synthesis of peptides was carried out on a 0.25 mmol scale using theFastMoc protocol (Applied Biosystems Inc). In each cycle of thisprotocol, 1.0 mmol of a dry protected amino acid in a cartridge wasdissolved in a solution of 0.9 mmol of HBTU, 2 mmol of DIEA, and 0.9mmol of HOBt in DMF with additional NMP added. The peptides were madeusing 0.1 mmol of NovaSyn TGR (Rink amide) resin (resin substitution 0.2mmol/g). The coupling time in this protocol was 21 min. Fmocdeprotection was carried out with 20% piperidine in NMP. At the end ofthe last cycle, the synthesized peptide was acetylated using aceticanhydride/DIEA/HOBt/NMP. The peptide resin was washed and dried forfurther manipulations or cleaved from the resin (using reagent B).Generally, the cleaved peptides were cyclized as in Method 1 beforepurification.

Method 6 Biotinylation of Resin-Bound Peptides

The peptides were prepared using Method 5. The ivDde protecting group onthe C-terminal lysine was selectively removed by treatment with 10%hydrazine in DMF. The resin was then treated with a solution ofBiotin-N-hydroxysuccinimidyl ester in DMF in the presence of DIEA. Afterwashing, the resin was dried and cleavage was performed using Reagent B.The resin was filtered off and the filtrate concentrated to dryness. Thebiotinylated peptide was dissolved in neat DMSO and treated with DIEAand stirred for 4-6 hours to effect disulfide cyclization. The crudemixture was purified by preparative HPLC.

In a typical experiment, 200 mg of the resin-bound peptide was treatedwith 10% hydrazine in DMF (2×20 mL) and washed with DMF (2×20 mL) andthen with dichloromethane (1×20 mL). The resin was resuspended in DMF(10 mL) and treated with a solution of Biotin-NHS ester (0.2 mmol, 5equivalents) and DIEA (0.2 mmol), and the resin was mixed with thereagents for 4 h. The completion of the reaction was checked by theninhydrin test. The peptide was then released from the resin bytreatment with Reagent B (10 mL) for 4 h. The resin was filtered off,Reagent B was removed in vacuo and the peptide was precipitated byaddition of anhydrous ether. The solid formed was collected, washed withether and dried. The solid was dissolved in anhydrous DMSO and themixture was adjusted to pH 7.5 with DIEA and stirred for 4-6 h to effectdisulfide cyclization. The disulfide cyclization reaction was monitoredby analytical HPLC. After completion of the cyclization, the mixturesolution was diluted with 25% acetonitrile in water and directlypurified by HPLC on a reverse phase C18 column using a gradient ofacetonitrile into water (both containing 0.1% TFA). Fractions wereanalyzed by analytical HPLC and those containing the pure product werecollected and lyophilized to obtain the required biotinylated peptide.

Method 7 Biotinylation of Purified Peptides

The purified peptide (10 mg, prepared by methods 1-5) containing a freeamino group was dissolved in anhydrous DMF or DMSO (1 mL) and Biotin-NHSester (5 equivalents) and DIEA (5 equivalents) were added. The reactionwas monitored by HPLC and after the completion of the reaction (1-2 h.),the crude reaction mixture was directly purified by preparative HPLC.Fractions were analyzed by analytical HPLC, and those containing thepure product were collected and lyophilized to obtain the requiredbiotinylated peptide.

Method 8 Biotinylation of Resin-Bound Peptides Containing Linkers

In a typical experiment, 400 mg of the resin-containing peptide (madeusing the ABI 433A Synthesizer and bearing an ivDde-protected lysine)was treated with 10% hydrazine in DMF (2×20 mL). The resin was washedwith DMF (2×20 mL) and DCM (1×20 mL). The resin was resuspended in DMF(10 mL) and treated with Fmoc-aminodioxaoctanoic acid (0.4 mmol), HOBt(0.4 mmol), DIC (0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. Afterthe reaction, the resin was washed with DMF (2×10 mL) and with DCM (1×10mL). The resin was then treated with 20% piperidine in DMF (2×15 mL) for10 min. each time. The resin was washed and the coupling withFmoc-diaminodioxaoctanoic acid and removal of the Fmoc protecting groupwere repeated once more. The resulting resin, containing a peptide witha free amino group, was treated with a solution of Biotin-NHS ester (0.4mmol, 5 equivalents) and DIEA (0.4 mmol, 5 equivalents) in DMF for 2hours. The peptide-resin was washed and dried as described previouslyand then treated with reagent B (20 mL) for 4 h. The mixture wasfiltered, and the filtrate concentrated to dryness. The residue wasstirred with ether to produce a solid that was collected, washed withether and dried. The solid was dissolved in anhydrous DMSO and the pHadjusted to 7.5 with DIEA. The mixture was stirred for 4-6 hr to effectthe disulfide cyclization reaction, which was monitored by analyticalHPLC. After the completion of the cyclization, the DMSO solution wasdiluted with 25% acetonitrile in water and applied directly to a reversephase C-18 column. Purification was effected using a gradient ofacetonitrile into water (both containing 0.1% TFA). Fractions wereanalyzed by analytical HPLC, and those containing the pure product werecollected and lyophilized to provide the required biotinylated peptide.

Method 9 Formation of 5-Carboxyfluorescein-Labeled Peptides

Peptide-resin obtained via Method 5, containing an ivDde protectinggroup on the epsilon nitrogen of lysine, was mixed with a solution ofhydrazine in DMF (10% hydrazine/DMF, 2×10 mL, 10 min) to remove theivDde group. The epsilon nitrogen of the lysine was labeled withfluorescein-5-isothiocyanate (0.12 mmol) and diisopropylethylamine (0.12mmol) in DMF. The mixture was agitated for 12 h (fluorescein-containingcompounds were protected from light). The resin was then washed with DMF(3×10 mL) and twice with CH₂Cl₂ (10 mL) and dried under nitrogen for 1h. The peptide was cleaved from the resin using reagent B for 4 h andthe solution collected by filtration. The volatiles were removed underreduced pressure, and the residue was dried under vacuum. The peptidewas precipitated with ether, collected and the precipitate was driedunder a stream of nitrogen. The precipitate was added to water (1 mg/mL)and the pH of the mixture was adjusted to 8 with 10% aqueous meglumine.Cyclization of the peptide was carried out for 48 h and the solution wasfreeze-dried. The crude cyclic peptide was dissolved in water andpurified by RP-HPLC on a C₁₈ column with a linear gradient ofacetonitrile into water (both phases contained 0.1% TFA). Fractionscontaining the pure product were collected and freeze-dried. Thepeptides were characterized by ES-MS and the purity was determined byRP-HPLC (linear gradient of acetonitrile into water/0.1% TFA).

Method 10A Preparation of Peptidic Chelate for Binding to Tc by Couplingof Single Amino Acids

Peptides were synthesized starting with 0.1 mmol of NovaSyn-TGR resin(0.2 mmol/g substitution). Deprotected (ivDde) resin was then treatedaccording to the protocol A for the incorporation of Fmoc-Gly-OH,Fmoc-Cys(Acm)-OH and Fmoc-Ser(tBu)-OH.

Protocol A for manual coupling of single amino acid:

1. Treat with 4 equivalents of corresponding Fmoc-amino acid and 4.1equivalents of HOBt and 4.1 equivalents of DIC for 5 h.

2. Wash with DMF (3×10 mL)

3. Treat with 20% piperidine in DMF (2×10 mL, 10 min.)

4. Wash with DMF (3×10 mL)

The Fmoc-protected peptide loaded resin was then treated with 20%piperidine in DMF (2×10 mL, 10 min.) and washed with DMF (3×10 mL). Asolution of N,N-dimethylglycine (0.11 mmol), HATU (1 mmol), and DIEA(0.11 mmol) in DMF (10 mL) was then added to the peptide loaded resinand the manual coupling was continued for 5 h. After the reaction theresin was washed with DMF (3×10 mL) and CH₂Cl₂ (3×10 mL) and dried undervacuum.

Method 10B Preparation of Peptidic Chelate for Binding to Tc byAppendage of the Glutaryl-PnAO6 Chelator to the Peptide Preparation of4-{2-(2-Hydroxyimino-1,1-dimethylpropylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyricacid, N-hydroxysuccinimide ester (Compound B, FIG. 86)

4-{2-(2-Hydroxyimino-1,1-dimethyl-propylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyricacid (Compound A, FIG. 86) (40 mg) was dissolved in DMF (700 μL).N-Hydroxysuccinimide (1.5 equiv, 17.2 mg) and1,3-diisopropylcarbodiimide (1.5 equiv, 24 μL) were added. The progressof the reaction was monitored by mass spectroscopy. After 17 h, thereaction was complete. The volatiles were removed in vacuo and theresidue was washed with ether (5×) to remove the unreacted NHS. Theresidue was dried to provide compound B, which was used directly withoutfurther treatment or purification. See FIG. 86 for reaction scheme.

Functionalization of Peptides with4-{2-(2-Hydroxyimino-1,1-dimethylpropylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyricacid, N-hydroxysuccinimide ester—(Compound B)

The peptide (prepared, for example, by Methods 1-13) is dissolved in DMFand treated with compound B and DIEA sufficient to maintain the basicityof the mixture. The progress of the reaction is monitored by HPLC andmass spectroscopy. At completion of the reaction the volatiles areremoved in vacuo and the residue is either purified by reverse phaseHPLC or processed further by selective removal of side chain protectinggroups or subjected to cleavage of all remaining protecting groups asrequired by the next steps in the synthesis scheme.

Method 11 Formation of Mercaptoacetylated Peptides UsingS-Acetylthioglycolic acid N-Hydroxysuccinimide Ester

S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA) (0.0055mmol) was added to a solution of a peptide (0.005 mmol, obtained fromMethods 1-5 with a free amine) in DMF (0.25 mL) and the reaction mixturewas stirred at ambient temperature for 6 h. The volatiles were removedunder vacuum and the residue was purified by preparative HPLC usingacetonitrile-water containing 0.1% TFA. Fractions containing the pureproduct were collected and freeze-dried to yield the mercaptoacetylatedpeptide. The mercaptoacetylated peptide was characterized by ESI-MS andthe purity was determined by reverse phase HPLC analysis employing alinear gradient of acetonitrile into water (both containing 0.1% TFA).

Examples of SATA-modified peptides include, but are not limited to:

SATA-modified SEQ ID NO: 480 Ac-AGPTWCEDDWYYCWLFGTGGGGK(SATA-JJ)-NH₂SATA-modified SEQ ID NO: 356 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SATA)-NH₂SATA-modified SEQ ID NO: 356Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SATA-JJ)-NH₂

Method 12 Formation of Mercaptoacetylated Peptides usingS-Acetylthioglycolic Acid

Purified peptides from method 5, after disulfide cyclization, wascoupled with S-acetylthioglycolic acid (1.5-10 eq.)/HOBt (1.5-10eq.)/DIC (1.5-10 eq.) in NMP for 2-16 hours at room temperature. Themixture was then purified by preparative HPLC; the fractions containingpure peptide were combined and lyophilized. In the case of compoundswith another lysine protected by an ivDde group, the deprotectionreaction employed 2% hydrazine in DMSO for 3 h at room temperature.Purification of the reaction mixture afforded pure peptide.

In the case when preparing a compound with S-acetylthioglycolic acidcoupled to two aminodioxaoctanoic acid groups and the peptide, thepurified peptide from method 5 (having a free amino group), was coupledto AcSCH₂CO—(NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO)₂—OH (30 eq.)/HOBt (30eq.)/DIC (30 eq.) in NMP for 40 hours at room temperature. The mixturewas purified, and the ivDde group was removed. A second purificationgave the final product as a white lyophilizate.

Alternatively Fmoc aminodioxaoctanoic acid was coupled twicesuccessively to the peptide (produced by method 5) followed by Fmocremoval and coupling to S-acetylthioglycolic acid.

Method 13 Preparation of Homodimers and Heterodimers

The required purified peptides were prepared by SPPS using Method 5. Toprepare homodimers, half of the peptide needed to prepare the dimer wasdissolved in DMF and treated with 10 equivalents of glutaric acid bisN-hydroxysuccinimidyl ester. The progress of the reaction was monitoredby HPLC analysis and mass spectroscopy. At completion of the reaction,the volatiles were removed in vacuo and the residue was washed withethyl acetate to remove unreacted bis-NHS ester. The residue was dried,re-dissolved in anhydrous DMF and treated with another half portion ofthe peptide in the presence of 2 equivalents of DIEA. The reaction wasallowed to proceed for 24 h. This mixture was applied directly to aWaters Associates C-18 XTerra reverse phase HPLC column and purified byelution with a linear gradient of acetonitrile into water (bothcontaining 0.1% TFA).

In the case of heterodimers, one of the monomers was reacted with thebis NHS ester of glutaric acid and after washing off the excess of bisNHS ester, the second peptide was added in the presence of DIEA. Afterthe reaction, the mixture was purified by preparative HPLC.

Example 1 Library Screening Against KDR and KDR/VEGF Complex Targets

Chimeric fusions of Ig Fc region with human KDR (#357-KD-050), murineKDR (#443-KD-050), human VEGFR-1 (#321-FL-050), human VEGFR-3(#349-F4-050), and human Trail R4 (#633-TR-100) were purchased incarrier-free form (no BSA) from R & D Systems (Minneapolis, Minn.).Trail R4 Fc is an irrelevant Fc fusion protein with the same Fc fusionregion as the target Fc fusion (KDR Fc) and is used to deplete thelibraries of Fc binders. VEGF₁₆₅ (#100-20) was purchased in carrier-freeform from Peprotech (Rocky Hill, N.J.). Protein A Magnetic Beads(#100.02) were purchased from Dynal (Oslo, Norway). Heparin (#H-3393)was purchased from Sigma Chemical Company (St. Louis, Mo.). A2-component tetramethyl benzidine (TMB) system was purchased from KPL(Gaithersburg, Md.).

In the following procedures, microtiter plates were washed with aBio-Tek 404 plate washer (Winooski, Vt.). ELISA signals were read with aBio-Tek plate reader (Winooski, Vt.). Agitation of 96-well plates was ona LabQuake shaker (Labindustries, Berkeley, Calif.).

Eight M13 phage display libraries were prepared for screening againstimmobilized KDR and VEGF/KDR targets: Cyclic peptide display librariesTN6/VI, TN7/IV, TN8/IX, TN9/IV, TN10/IX, TN12/I, and MTN13/I, and alinear display library, Lin20. The design of these libraries has beendescribed, supra.

The DNA encoding the library was synthesized with constant DNA on eitherside so that the DNA can be PCR amplified using Taq DNA polymerase(Perkin-Elmer, Wellesley, Mass.), cleaved with NcoI and PstI, andligated to similarly cleaved phage display vector. XL1-Blue MFR′ E. colicells were transformed with the ligated DNA. All of the libraries wereconstructed in same manner.

KDR Selection Protocol in the Presence of Heparin

Protein A Magnetic Beads were blocked once with 1×PBS (pH 7.5), 0.01%Tween-20, 0.1% HSA (Blocking Buffer) for 30 minutes at room temperatureand then washed five times with 1×PBS (pH 7.5), 0.01% Tween-20, 5 μg/mLheparin (PBSTH Buffer).

The cyclic peptide, or “constrained loop”, libraries were pooled for theinitial screening into two pools: TN6/VI, TN7/IV and TN8/IX were in onepool; TN9/IV, TN10/1× and TN12/I were in the second pool. The two pooledlibraries and the linear library (Lin20) were depleted against Trail R4Fc fusion (an irrelevant Fc fusion) and then selected against KDR Fcfusion. 10¹¹ plaque forming units (pfu) from each library per 100 μLPBSTH were pooled together, e.g., 3 pooled libraries would result in atotal volume of ˜350 μl in PBSTH.

To prepare the irrelevant Fc fusion beads, 500 μl of Trail R4-Fc fusion(0.1 μg/μl stock in PBST (no heparin)) were added to 1000 μl of washed,blocked protein A magnetic beads. The fusion was allowed to bind to thebeads overnight with agitation at 4° C. The next day, the magnetic beadswere washed 5 times with PBSTH. Each phage pool was incubated with 50 μlof Trail R4 Fc fusion beads on a Labquake shaker for 1 hour at roomtemperature (RT). After incubation, the phage supernatant was removedand incubated with another 50 μL of Trail R4 beads. This was repeatedfor a total of 5 rounds of depletion, to remove non-specific Fc fusionand bead binding phage from the libraries.

To prepare the KDR target beads, 500 μl of KDR-Fc fusion (0.1 μg/μlstock in PBST (no heparin)) were added to 500 μL of washed, blockedbeads. The KDR-Fc fusion was allowed to bind overnight with agitation at4° C. The next day, the beads were washed 5 times with PBSTH. Eachdepleted library pool was added to 100 μL of KDR-Fc beads and allowed toincubate on a LabQuake shaker for 1 hour at RT. Beads were then washedas rapidly as possible with 5×1 mL PBSTH using a magnetic stand(Promega) to separate the beads from the wash buffer. Phage still boundto beads after the washing were eluted once with 250 μl of VEGF (50μg/mL, ˜1 μM) in PBSTH for 1 hour at RT on a LabQuake shaker. The 1-hourelution was removed and saved. After the first elution, the beads wereincubated again with 250 μl of VEGF (50 μg/mL, ˜1 μM) overnight at RT ona LabQuake shaker. The two VEGF elutions were kept separate and a smallaliquot taken from each for titering. Each elution was mixed with analiquot of XL1-Blue MRF′ (or other F′ cell line) E. coli cells that hadbeen chilled on ice after having been grown to mid-logarithmic phase.The remaining beads after VEGF elution were also mixed with cells toamplify the phage still bound to the beads, i.e., KDR-binding phage thathad not been competed off by the two VEGF incubations (1-hour andovernight (O/N) elutions). After approximately 15 minutes at roomtemperature, the phage/cell mixtures were spread onto Bio-Assay Dishes(243×243×18 mm, Nalge Nunc) containing 250 mL of NZCYM agar with 50μg/mL of ampicillin. The plate was incubated overnight at 37° C. Thenext day, each amplified phage culture was harvested from its respectiveplate. Over the next day, the input, output and amplified phage cultureswere titered for FOI (i.e., Fraction of Input=phage output divided byphage input).

In the first round, each pool yielded three amplified eluates. Theseeluates were panned for 2-3 more additional rounds of selection using˜10¹⁰ input phage/round according to the same protocol as describedabove. For each additional round, the KDR-Fc beads were prepared thenight before the round was initiated. For the elution step in subsequentrounds, the amplified elution re-screen on KDR-Fc beads was alwayseluted in the same manner, and all other elutions were treated aswashes. For example, for the amplified elution recovered by using thestill-bound beads to infect E. coli, the 1-hour and overnight VEGFelutions were performed and then discarded as washes. Then the beadswere used to again infect E. coli and produce the next round amplifiedelution. Using this procedure, each library pool only yielded threefinal elutions at the end of the selection. Two pools and one linearlibrary, therefore, yielded a total of 9 final elutions at the end ofthe selection.

This selection procedure was repeated for all libraries in the absenceof heparin in all binding buffers, i.e., substituting PBST (PBS (pH7.5), 0.01% Tween-20) for PBSTH in all steps.

KDR Selection Protocol in the Absence of Heparin

++A true TN11/1 library was used to screen for KDR binders. The sameselection protocol as above (KDR Selection Protocol in the Presence ofHeparin) was used, except heparin was omitted. The three elutionconditions were VEGF elution (1 uM; 1 hr; same as original protocol),Dimer D6 elution (0.1 uM; 1 hr), and then bead elution (same as above).TN11/1 alone was used in the selection and screening. For selectedpeptides, see Table 27 and Consensus Sequence 9A.

KDR:VEGF Complex Selection Protocol in the Presence of Heparin

Protein A magnetic beads were blocked once with Blocking Buffer for 30minutes at room temperature and then washed five times with PBSTH.

Two pools of constrained loop libraries and a linear library (Lin20)were prepared as before and then depleted against KDR Fc fusion alone,instead of Trail-R4 Fc fusion, to remove binders to the receptor withoutbound VEGF. Once depleted, the libraries were selected against theKDR:VEGF₁₆₅ complex.

To prepare KDR-Fc fusion depletion beads, 1 mL of KDR-Fc fusion (0.1μg/μL stock in PBST (no heparin)) was added to 1 mL of washed, blockedbeads. The fusion was allowed to bind overnight with agitation at 4° C.The next day, the beads were washed 5 times with PBSTH. Each phage poolwas incubated with 50 μl of KDR-Fc fusion beads on a LabQuake shaker for1 hour at RT. After incubation, the phage supernatant was removed andincubated with another 50 μL of KDR-Fc beads. This was repeated for atotal of 5 rounds of depletion.

To prepare the KDR:VEGF complex beads, 300 μL of KDR-Fc fusion beadsfrom above were incubated with 15 μL of VEGF (1 mg/mL). VEGF was allowedto bind for 1 hour at RT. The beads were washed 5 times with PBSTH. Eachdepleted library pool was added to 100 μl of KDR:VEGF complex beads andallowed to incubate on a LabQuake shaker for 1 hour at RT. Beads werethen washed as rapidly as possible with 5×1 mL PBSTH using a magneticstand (Promega) to separate the beads from the wash buffer. To elute thephage still bound after washing, the beads were mixed with cells toamplify the phage still bound to the beads. After approximately 15minutes at room temperature, the phage/cell mixtures were spread ontoBio-Assay Dishes (243×243×18 mm, Nalge Nunc) containing 250 mL of NZCYMagar with 50 μg/mL of ampicillin. The plate was incubated overnight at37° C. The next day, each amplified phage culture was harvested from itsrespective plate. Over the next day, the input, output and amplifiedphage cultures were titered for FOI. This selection protocol wasrepeated for two additional rounds using 10¹⁰ input phage from eachamplified elution.

KDR and KDR/VEGF Screening Assay

100 μl of KDR-Fc fusion or Trail R4-Fc fusion (1 μg/mL) were added toduplicate Immulon II plates, to every well, and allowed to incubate at4° C. overnight. Each plate was washed twice with PBST (PBS, 0.05%Tween-20). The wells were filled to the top with 1×PBS, 1% BSA andallowed to incubate at RT for 2 hours. Each plate was washed once withPBST (PBS, 0.05% Tween-20).

To assess binding to KDR:VEGF complex, another set of KDR plates wasprepared as above and then 100 μL of VEGF (1 μg/mL) in PBST was added toeach KDR well and allowed to incubate at RT for 30 minutes. Each platewas then washed with PBST (PBS, 0.05% Tween-20).

Once the plates were prepared, each overnight phage culture was diluted1:1 (or to 10¹⁰ pfu if using purified phage stock) with PBS, 0.05%Tween-20, 1% BSA. 100 μl of each diluted culture was added and allowedto incubate at RT for 2-3 hours. Each plate was washed 5 times withPBST. The binding phage were visualized by adding 100 μl of a 1:10,000dilution of HRP-anti-M13 antibody conjugate (Pharmacia), diluted inPBST, to each well, then incubating at room temperature for 1 hr. Eachplate was washed 7 times with PBST (PBS, 0.05% Tween-20), then theplates were developed with HRP substrate (˜10 minutes) and theabsorbance signal (630 nm) detected with plate reader.

KDR and VEGF/KDR complex binding phage were recovered, amplified, andthe sequences of the display peptides responsible for the binding weredetermined by standard DNA sequencing methods. The binding peptides ofthe phage isolates are set forth in Tables 1-7, infra.

After isolation of KDR and VEGF/KDR complex isolates in initialselection rounds, certain isolates were selected to act as templates forthe construction of secondary libraries, from which additional highaffinity binding polypeptides were isolated. In a secondary TN8 library,the phage isolate sequence PKWCEEDWYYCMIT (SEQ ID NO: 21) was used as atemplate to construct a library that allowed one-, two-, and three-basemutations to the parent sequence at each variable codon. In a secondaryTN12 library, the phage isolate sequence SRVCWEDSWGGEVCFRY (SEQ ID NO:88) was used as a template to construct a library that allowed one-,two-, and three-base mutations to the parent sequence at each variablecodon. In a another TN8 secondary library, a recurrent motif from theinitial TN8 sequences was kept constant (WVEC---TG-C---; SEQ ID NO: 260)and all of the other codon positions (i.e., at “-”) were allowed to vary(all possible 20 amino acids) using NNK codon substitution, where Nstands for any nucleotide and K stands for any keto nucleotide (G or T).

Using a method of peptide optimization by soft randomization asdescribed by Fairbrother et al., Biochemistry, 37(51):17754-17764(1998), two libraries were prepared based on the SEQ ID NO: 21 and SEQID NO: 88 sequences. At each residue position, each nucleotide within aparticular codon was allowed to evolve by adding fixed amounts of theother three nucleotides that did not correspond to the nucleotide of theparent codon. This nucleotide mixing is accomplished in the synthesis ofthe template DNA used to make the library. For these libraries, theparent nucleotide within each codon was maintained at 64% for SEQ ID NO:21 and 67% for SEQ ID NO: 88, whereas the other nucleotides were addedat the remainder frequency divided by three. Since the parentnucleotides are in the majority, the overall consensus sequence for thewhole library should still contain the parental sequence. Inspection ofindividual isolates, however, shows that multiple mutations arepossible, thus allowing selection of peptides with improved bindingability compared to the parent sequence.

For the third library, the TN8 motif described above was kept constantand all of the other positions in were allowed to vary with NNKsubstitution in the template oligonucleotide. To extend thesubstitution, NNK diversity was also permitted in the two flanking aminoacid positions, thus adding variable amino acid positions N-terminal andC-terminal to the display peptide. The secondary library template,therefore, encoded a display peptide of the following sequence:Xaa-Xaa-Trp-Val-Glu-Cys-Xaa-Xaa-Xaa-Thr-Gly-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa(SEQ ID NO: 261), where Xaa can be any amino acid. Unlike the previoustwo libraries, where the consensus sequence remains the parentalsequence, this library was quite diverse in all allowed positions andonly resembled the parent motif in the residues that were held constant.

A total of 2×10¹¹ pfu from each library was used as before, except theelution strategy was changed. Competition elution of bound phage wasperformed using the parental peptide (50 μM) that was used to make theparticular secondary library (i.e., peptides of SEQ ID NOS: 21, 88, and40, respectively). Binding phage were eluted through three steps: (1)elution for 1 hour at room temperature, the eluted phage being used toinfect cells for amplification, (2) elution overnight, wherein freshcompetition elution peptide was added to the bound phage and incubatedat 4° C. overnight with mixing, the eluted phage being then used toinfect cells for amplification, and (3) the remaining beads (bearinguneluted binding phage) were used to infect cells directly. Three roundsof selections were performed. Plaques were picked from rounds 2 and 3and analyzed by ELISA and sequencing. KDR positive isolates were assayedfurther for competition with 50 μM free parent peptide. Those peptidesthat showed minimal competition with the parent peptide were deemedhigher affinity binders and were synthesized. These sequences are listedin the following table as SEQ ID NOS: 22-33 for the TN8 secondarylibrary and SEQ ID NOS: 89-95 for the TN12 secondary library.

TABLE 1 TN8/IXLibrary Isolates Sequence SEQ ID NO: Elution ClassDSWCSTEYTYCEMI 20 1 HR NA PKWCEEDWYYCMIT 21 1 HR (III) SDWCRVDWYYCWLM 22O/N III ANWCEEDWYYCFIT 23 O/N III ANWCEEDWYYCWIT 24 O/N IIIPDWCEEDWYYCWIT 25 O/N III SNWCEEDWYYCYIT 26 O/N III PDWCAADWYYCYIT 27O/N III PEWCEVDWYYCWLL 28 CELL III PTWCEDDWYYCWLF 29 O/N IIISKWCEQDWYYCWLL 30 CELL III RNWCEEDWYYCFIT 31 O/N III VNWCEEDWYYCWIT 32O/N III ANWCEEDWYYCYIT 33 O/N III VWECAKTFPFCHWF 34 1 HR IVTVCYEGTRICEWH 35 1 HR NA WVECRYSTGLCINY 36 O/N NA WYWCDYYGIGCKWT 371 HR NA WVECWWKSGQCYEF 38 1 HR, CELL (II) WIQCDMETGLCTHG 39 1 HR, CELLII WVECFMDTGACYTF 40 CELL, O/N II WLECYAEFGHCYNF 41 CELL, O/N IIWIECDMLTGMCKHG 42 CELL NA SVECFMDTGACYTF 43 CELL I WIQCNSITGHCTSG 44CELL II WIECYHPDGICYHF 45 CELL (III) QAWVECYAETGYCWPRSW 46 NA NAVGWVECYQSTGFCYHSRD 47 NA NA FTWVECHQATGRCVEWTT 48 NA NADWWVECRVGTGLCYRYDT 49 NA NA DSWVECDAQTGFCYSFLY 50 NA NAGGWVECYWATGRCIEFAG 51 NA NA ERWVECRAETGFCYTWVS 52 NA NAGGWVECRAETGHCQEYRL 53 NA NA VAWVECYQTTGKCYTFRG 54 NA NAEGWVECFANTGACFTYPR 55 NA NA GVECYKHSGMCRSW 56 O/N II GVWCDMVTGWCYHG 57CELL II WIECHYKTGHCIHS 58 CELL II DFNCKMIDGFCLLK 59 1 HR IIWIQCDRKAGRCSRG 60 CELL II TITCWMDTGHCMHE 61 CELL II GINCYPATGKCQMG 62CELL II WTECHYATGKCHSF 63 CELL II LNICKEDWYYCFLL 64 1 HR I/IIIGITCYSATGKCQMW 65 CELL II WVQCASDTGKCIMG 66 CELL II TGNCQEDWYYCWYF 67CELL II KELCEDDWYYCYLM 68 1 HR I/III HWECYSDTGKCWFF 69 O/N IIGITCYSDTGKCFSF 70 CELL II AVTCWALTGHCVEE 71 O/N II YVDCYYDTGRCYHQ 72CELL II WYWCQYHGVCPQS* 73 1 HR I/III LVMCISPEGYCYEI 74 O/N IILIECYAHTGLCFDF 75 O/N II HWWCAFQPQECEYW 76 1 HR III HYECWYPEGKCYFY 77CELL II WYWCHHIGMYCDGF 78 1 HR III WEWCPIDAWECIML 79 1 HR IIWLECYTEFGHCYNF 80 1 HR II WVECWWKYGQCYEF 81 1 HR II PNTCETFDLYCWWI 821 HR II WIICDGNLGWCWEG 83 O/N II GEQCSNLAVACCST 84 O/N II WVECYDPWGWCWEW85 CELL NA WYWCMHYGLGCPYR 86 CELL NA

TABLE 2 TN12/I Library Isolates* SEQ ID Sequence NO: Elution ClassYPWCHELSDSVTRFCVPW 87 1 HR (III) SRVCWEDSWGGEVCFRY 88 1 HR (III)SRVCWEYSWGGEVCYRV 89 O/N III FGECWEYFWGGEFCLRV 90 CELL IIIWRICWESSWGGEVCIGH 91 CELL III YGVCWEYSWGGEVCLRF 92 CELL IIISSVCFEYSWGGEVCFRY 93 CELL III SRVCWEYSWGGQICLGY 94 CELL IIIFSVCWEYSWGGEVCLRQ 95 CELL III DHMCRSPDYQDHVFCMYW 96 CELL (II)PPLCYFVGTQEWHHCNPF 97 CELL (II) WWECKREEYRNTTWCAWA 98 CELL IIDSYCMMNEKGWWNCYLY 99 CELL NA PAQCWESNYQGIFFCDNP 100 CELL II?GSWCEMRQDVGKWNCFSD 101 CELL II GWACAKWPWGGEICQPS 102 CELL (II)ASTCVFHDHPYFPMCQDN 103 CELL I/III PDTCTMWGDSGRWYCFPA 104 CELL (II)NWKCEYTQGYDYTECVYL 105 O/N II NWECGWSNMFQKEFCARP 106 1 HR (III)SGYCEFESDTGRWFCSSW 107 O/N II GGWCQLVDHSWWWCGDS 108 O/N IIDNWCEIVVEKGQWFCYGS 109 O/N II YPGCYETSLSGVWFCADG 110 CELL IIGWCQMDAQGIWSCWAD 111 1 HR II DRWCMLDQEKGWWLCGPP 112 CELL IINSECGCPNMLHKEFCARH 113 1 HR I/III PFWCKFQQSKAMFPCSWF 114 1 HR IIYPWCHEHSDSVTRFCVPW 115 1 HR III SDLCYNQSGWWELCYFD 116 O/N I/II?LGYCMYDYENRGWTCYPP 117 O/N II YYQCQRYWDGKTWWCEYN 118 1 HR I/IIIDSWCELEHQSGIWRCDFW 119 CELL II DWACDEYWSAYSVLCKHP 120 CELL IILSLCYNDMHGWWEHCQWY 121 CELL II YSHCIETSMENIWFCDFD 122 CELL IIPPFCIYQEPSGQWWCYDH 123 CELL II PGWCDFSPQLGQWMCDWF 124 CELL IILDNCIWNVWKGVQDCEYS 125 O/N II AGWCEYVAPQGAWRCFHN 126 CELL IIWDDCIWHMWLKKKDCNSG 127 O/N II PGHCEYIWIDEQPWCVRL 128 CELL IIIYSDCLFQLWKGSVCPPS 129 CELL II YFFCSFADVAYESCHPL 130 CELL NANYMCESEDHTYMFPCWWY 131 CELL NA DAVCYNPWFKYWETCEYN 132 CELL NANYMCEYEDHTYMLTCECN 133 CELL NA WDDCIYSMWMVHTVCDR 134 CELL NANWKCDAHQEGRIHICWGY 135 CELL NA NGSCWYDFGWETEICFHN 136 CELL II

TABLE 3 Lin20 Library Isolates* Sequence SEQ ID NO: Elution ClassQVQYQFFLGTPRYEQWDLDK 137 CELL II EPEGYAYWEVITLYHEEDGD 138 CELL (II)WYYDWFHNQRKPPSDWIDNL 139 1 HR III AFPRFGGDDYWIQQYLRYTD 140 1 HR (III)GDYVYWEIIELTGATDHTPP 141 O/N (III) RGDYQEQYWHQQLVEQLKLL 142 1 HR (III)RSWYLGPPYYEEWDPIPN 143 CELL II PSNSWAAVWEDDMQRLMRQH 144 CELL IIPRLGDDFEEAPPLEWWWAHF 145 CELL II MPPGFSYWEQVVLHDDAQVL 146 CELL IIKKEDAQQWYWTDYVPSYLYR 147 1 HR III? WVTKQQFIDTYGRKEWTILF 148 CELL IIWLYDYWDRQQKSEEFKFWSQ 149 1 HR III PVTDWTPHHPKAPDVWLFYT 150 1 HR III?EWYWTEHVGMKHGFFV 151 1 HR I/III DALEAPKRDWYYDWFLNHSP 152 1 HR IIIPDNWKEFYESGWKYPSLYKPL 153 1 HR NA EWDAQYWHDLRQQYMLDYIQ 154 1 HR I/IIIAFEIEYWDSVRNKIWQHFPD 155 1 HR I/III AFPRFGGDDYWIQQYLRYTF 156 1 HR I/IIIAHMPPWRPVAVDALFDWVE 157 CELL NA AHMPPWWPLAVDAQEDWFE 158 CELL NAAQMPPWWPLAVDALFDWFE 159 CELL II ARMGDDWEEAPPHEWGWADG 160 CELL IIDWYWQRERDKLREHYDDAFW 161 1 HR I/III DWYWREWMPMHAQFLADDW 162 1 HR I/IIIDWYYDEILSMADQLRHAFLS 163 1 HR III EEQQALYPGCEPAEHWVYAG 164 1 HR IIIFDVVNWGDGIWYAYPS 165 CELL II FPSQMWQQKVSHHFFQHKGY 166 CELL IIGSDHVRVDNYWWNGMAWEIF 167 1 HR II ISPWREMSGWGMPWITAVPH 168 1 HR I/IIILEEVFEDFQDFWYTEHIIVDR 169 1 HR II MPPGFSYWEQAALHDDAQDL 170 CELL IIPEDSEAWYWLNYRPTMFHQL 171 1 HR I/III? QIEYVNDKWYWTGGYWNVPF 172 1 HR IIQVQYQFILGTPRYEQWDPDK 173 CELL II RDEWGWTGVPYEGEMGYQIS 174 1 HR IISTNGDSFVYWEEVELVDHPY 175 O/N II SYEQWLPQYWAQYKSNYFL 176 1 HR I/III?TKWGPNPEHWQYWYSHYASS 177 1 HR I/III? VSKGSIDVGEGISYWEIIEL 178 1 HR IIIWESDYWDQMRQQLKTAYMKV 179 1 HR I/III WYHDGLHNERKPPSHWIDNV 180 1 HR IIIAPAWTFGTNWRSIQRVDSLT 181 CELL NA EGWFRNPQEIMGFGDSWDKP 182 CELL NAGWDLSVNRDKRWFWPWSSRE 183 CELL NA KSGVDAVGWHIPVWLKKYWF 184 CELL NAGMDLYQYWASDDYWGRHQEL 185 CELL NA GVDIWHYWKSSTRYFHQ 186 CELL NA

TABLE 4 TN7/IV Library Isolates Sequence SEQ ID NO: Elution ClassGVECNHMGLCVSW 187 CELL II GITCDELGRCVHW 188 CELL II WIQCNHQGQCFHG 189CELL II WIECNKDGKCWHY 190 CELL II WVECNHKGLCREY 191 CELL IIWYWCEFYGVCSEE 192 1 HR I/III

TABLE 5 TN9/IV Library Isolates SEQ ID Sequence NO: Elution ClassIDFCKGMAPWLCADM 193 1 HR (III) PWTCWLEDHLACAML 194 CELL IIDWGCSLGNWYWCSTE 195 CELL NA MPWCSEVTWGWCKLN 196 CELL II RGPCSGQPWHLCYYQ197 O/N II PWGCDHFGWAWCKGM 198 O/N NA MPWCVEKDHWDCWWW 199 CELL NAPGPCKGYMPHQCWYM 200 CELL NA YGPCAEMSPWLCWYP 201 CELL NA YGPCKNMPPWMCWHE202 CELL NA GHPCKGMLPHTCWYE 203 CELL NA

TABLE 6 TN10/IX Library Isolates SEQ ID Sequence NO: Elution ClassNNSCWLSTTLGSCFFD 204 O/N NA DHHCYLHNGQWICYPF 205 CELL (III)NSHCYIWDGMWLCFPD 206 CELL (II)

TABLE 7 MTN13/I Library Isolates Sequence SEQ ID NO: Elution ClassSNKCDHYQSGPHGKICVNY 207 CELL NA SNKCDHYQSGPYGEVCFNY 208 CELL NARLDCDKVFSGPYGKVCVSY 209 CELL NA RLDCDKVFSGPDTSCGSQ 210 CELL NARLDCDKVFSGPHGKICVRY 211 CELL NA RLDCDKVFSGPHGKICVNY 212 CELL NARVDCDKVISGPHGKICVNY 213 CELL NA RTTCHHQISGPHGKICVNY 214 CELL NAEFHCHHIMSGPHGKICVNY 215 CELL NA HNRCDFKMSGPHGKICVNY 216 CELL NAWQECTKVLSGPGTFECSYE 217 CELL NA WQECTKVLSGPGQFSCVYG 218 CELL NAWQECTKVLSGPGQFECEYM 219 CELL NA WQECTKVLSGPNSFECKYD 220 CELL NAWDRCERQISGPGQFSCVYG 221 CELL NA WQECTKVLSGPGQFLCSYG 222 CELL NARLDCDMVFSGPHGKICVNY 223 CELL NA KRCDTTHSGPHGIVCVVY 224 CELL NASNKCDHYQSGPYGAVCLHY 225 CELL NA SPHCQYKISGPFGPVCVNY 226 CELL NAAHQCHHWTSGPYGEVCFNY 227 CELL NA YDKCSSRFSGPFGEICVNY 228 CELL NAMGGCDFSFSGPFGQICGRY 229 CELL NA RTTCHHQISGPFGDVCVSY 230 CELL NAWYRCDFNMSGPDFTECLYP 231 CELL NA WMQCNMSASGPKDMYCEYD 232 CELL NAGISCKWIWSGPDRWKCHHF 233 CELL NA WQVCKPYVSGPAAFSCKYE 234 CELL NAGWWCYRNDSGPKPFHCRIK 235 CELL NA EGWCWFIDSGPWKTWCEKQ 236 CELL NAFPKCKFDFSGPPWYQCNTK 237 CELL NA RLDCDKVFSGPYGRVCVKY 238 CELL NARLDCDKVFSGPYGNVCVNY 239 CELL NA RLDCDKVFSGPSMGTCKLQ 240 CELL NARTTCHHHISGPHGKICVNY 241 CELL NA QFGCEHIMSGPHGKICVNY 242 CELL NAPVHCSHTISGPHGKICVNY 243 CELL NA SVTCHFQMSGPHGKICVNY 244 CELL NAPRGCQHMISGPHGKICVNY 245 CELL NA RTTCHHQISGPHGQICVNY 246 CELL NAWTICHMELSGPHGKICVNY 247 CELL NA FITCALWLSGPHGKICVNY 248 CELL NAMGGCDFSFSGPHGKICVNY 249 CELL NA KDWCHTTFSGPHGKICVNY 250 CELL NAAWGCDNMMSGPHGKICVNY 251 CELL NA SNKCDHIMSGPHGKICVNY 252 CELL NASNKCDHYQSGPFGDICVMY 253 CELL NA SNKCDHYQSGPFGDVCVSY 254 CELL NASNKCDHYQSGPFGDICVSY 255 CELL NA RTTCHHQISGPFGPVCVNY 256 CELL NARTTCHHQISGPYGDICVKY 257 CELL NA PHGKICVNYGSESADPSYIE 258 CELL NARYKCPRDLSGPPYGPCSPQ 259 CELL NA

TABLE 27 TN11.1 Library Isolates # of Sequence SEQ ID NO: Elutionisolates GSNMVCMDDSYGGTTCYSMAP 505 D6 107 GSYNQCYGDYWGGETCYLIAP 506 Bead93 GSRVNCGAEDGLSFLCMMDAP 507 Bead 40 GSIWDCQISEYGGEDCYLVAP 508 D6 29GSYWHCMDDFFGGETCFATAP 509 D6 28 GSGEYCFPSIYGGETCYAHAP 510 D6 24GSEQLCFEYQYGGVECFGPAP 511 D6 21 GSTGVCSPAPYGGEVCYHFAP 512 D6 20GSHDECWEDIYGGFTCMLMAP 513 D6 19 GSQHTCFSDPYGGEVCYADAP 514 D6 18GSWEVCENSNYGGQICYWFAP 515 D6 18 GSHEMCWSDVWGGLTCMTMAP 516 D6 15GSLSLCKFFGDGSYYCEPPAP 517 D6 14 GSTRFCEPYQWGGEVCYWKAP 518 D6 14GSFSTCATFPWTTKFCSNMAP 519 VEGF 12 GSHELCFEGTYGGEVCFSMAP 520 D6 12GSLWHCFNDVYGGENCIPFAP 521 VEGF 12 GSQQYCIPAEYGGMECYPFAP 522 Bead 11GSIQNCWKYEFGGIVCMDMAP 523 D6 9 GSVSGCKEFWNSSGRCFTHAP 524 D6 9GSLWECRGDFYGGEVCFNYAP 525 D6 8 GSNLICYDYYYGGQDCYHDAP 526 D6 8GSEGTCEEYQYGGIVCWWGAP 527 D6 7 PGSGDCDWYYEWLFDCPLNAP 528 VEGF 7GSDQMCFNESFGGQICFYSAP 529 VEGF 6 GSGMACMSDPYGGQVCYAIAP 530 D6 5GSELTCWDSAYGGNECFFFAP 531 VEGF 4 GSHFLCVKEMEGGETCYYSAP 532 VEGF 4GSWEICFAGPYGGSWCIPEAP 533 Bead 4 GSAQYCMESYYGGFTCVTLAP 534 Bead 3GSFNACGFEEGLEWMCYRQAP 535 D6 3 GSKLLCQYWEHEWWPCMNEAP 536 VEGF 3GSNMNCGAEQGLESLCGWRAP 537 VEGF 3 GSNWVCLSEGYGGMTCYPSAP 538 VEGF 3GSPSTCIYSSGLIVDCGLLAP 539 VEGF 3 GSTQHCWPSEYGGMTCVPAAP 540 D6/VEGF 3GSTWACEEISAHHTKCTYQAP 541 VEGF/Bead 3 GSYTECWEEDYGGVTCFNVAP 542 Bead 3GSDKFCFKDPWGGVTCYHLAP 543 D6 2 GSDLDCWTDPYGGEVCYWHAP 544 D6 2GSDYECYNAWFGYFDCPGDAP 545 VEGF/Bead 2 GSLSTCWKQAYGGVWCVDHAP 546 VEGF 2GSMQLCRQWAYGGQTCYWYAP 547 D6 2 GSNQLCITAQFGGQDCYPIAP 548 VEGF 2GSPMWCAPWPWGGEHCVGSAP 549 VEGF 2 GSQLLCGSEPELAWMCEQGAP 550 VEGF 2GSQRQCWDDYFGGIICYVIDA 551 VEGF 2 GSREVCWQDFFGGMVCVRDAP 552 Bead 2GSSQWCQRDFWGGDICINLAP 553 VEGF 2 GSTDICWPGSYGGEICIPRAP 554 VEGF 2GSTEYCWPEPHGGQACILLAP 555 VEGF 2 GSTHFCIDYIWGGKHCIADAP 556 VEGF 2GSTMMCWPAHYGGDECFALAP 557 VEGF 2 GSTQMCFPHQYGGQSCYSFAP 558 VEGF 2GSVEGCWVEDQTSPFCWIDAP 559 VEGF 2 GSWYTCWDEASGGQVCYQLAP 560 VEGF 2GSYNLCYPEIYGGQVCYRMAP 561 D6 2 GSYSQCFPDPFGGTTCFVSAP 562 D6 2GSSMQCFNRVSQLVDCETAAP 563 VEGF 2 GSAKTCRSYWAQSGYCYEYAP 564 D6 1GSAQTCWDYVYGGFFCLNTAP 565 VEGF 1 GSAWDCFQQDTYSTHCHWRAP 566 VEGF 1GSAWNCEMLDPWSTQCSWDAP 567 VEGF 1 GSAWVCHPEQEGGTTCYWVAP 568 VEGF 1GSDELCWPQEFGGWVCIQGAP 569 Bead 1 GSDFQCFNWEGYPTNCYSNAP 570 D6 1GSDKKCWPSPYGGQICWAVAP 571 VEGF 1 GSDQLCFDQRWGGQVCVFGAP 572 VEGF 1GSDSGCKEFWNSSDRCYTHAP 573 D6 1 GSEWICWSSFFGGETCTPKAP 574 VEGF 1GSEWNCLNNTPYQTTCSWRAP 575 Bead 1 GSEWRCWPDVFGGQMCFNMAP 576 VEGF 1GSEYECYPDWYGGEVCVQKAP 577 VEGF 1 GSFEACWEEAYGGLTCWHDAP 578 D6 1GSFEECMPYRYGGQTCFMIAP 579 D6 1 GSFWTCVDTNWHTTECFHSAP 580 VEGF 1GSGQMCWHGQYGGTICVAMAP 581 VEGF 1 GSGWVCKQQGPHKTECLFMAP 582 VEGF 1GSHDECWEDIYGGFTCMPYGS 583 D6 1 GSHVVCWDDPYGGESCYNTAP 584 VEGF 1GSIDICTDSYWGGITCYKFAP 585 D6 1 GSKWICVDVKWGGSACYDIAP 586 VEGF 1GSLWECRIDYYGGEVCFIDAP 587 D6 1 GSLWTCVLSVYGGEDCYNLAP 588 VEGF 1GSMTMCGAEPDLWYMCYGIAP 589 VEGF 1 GSNQYCMPYDWGGEMCFEVAP 590 D6 1GSNVFCSEGPFGGEICYGIAP 591 VEGF 1 GSNWACFIEAMGGWTCAPRPT 592 VEGF 1GSNWTCFIDSFQGETCYPFAP 593 VEGF 1 GSNWWCHSEAFGGHTCYNAAP 594 VEGF 1GSPCACNNSYGHSDDCDHLAP 595 VEGF 1 GSPGNCKDFWAWSLQCFSFAP 596 VEGF 1GSPRWCYFSSGIMKDCDILAP 597 VEGF 1 GSPTYCQFHSGVVTLCSMFAP 598 VEGF 1GSQEICFNSQYGGQVCFDSAP 599 D6 1 GSQMICYPHVFGGQDCFPGAP 600 VEGF 1GSQWTCTELSDVMTHCSYTAP 601 VEGF 1 GSRVNCGAEDDLSFLCMTEAP 602 VEGF 1GSSGDCIEMYNDWYYCTILAP 603 Bead 1 GSSWECGEFGDTTIQCNWVAP 604 VEGF 1GSSWQCFSEAPSGATCVPIAP 605 VEGF 1 GSSWQCVQVDDFHTECSFMAP 606 VEGF 1GSSWTCVFYPYGGEVCIPDAP 607 D6 1 GSTELCVPYQWGGEVCVAQAP 608 D6 1GSTVYCHNEYFGGQVCFTIAP 609 VEGF 1 GSTYGCEYYMPFQHKCSVEAP 610 VEGF 1GSWWGCFPYSWGGEICTSIAP 611 D6 1 GSWWNCVDTSFHTTQCKYAAP 612 VEGF 1GSYFMCQDGFWGGQDCFYIAP 613 VEGF 1 GSYMWCTESKFGGSTCFNLAP 614 VEGF 1GSGAYSHLLEYHAVCKNVAP 615 VEGF 1 PGSWTCQNYEPWATTCVYDAP 616 VEGF 1

* During the course of DNA synthesis, there is always a small percentageof incomplete couplings at each cycle. Since the libraries used forthese experiments were constructed using TRIM technology to coupletrinucleotides (codons) instead of nucleotides, the library template DNAoften has a small percentage of deleted codons. In the case of the TN12library, for instance, it has been observed that approximately 5.3% ofthe total library is phage expressing a cyclic 11-mer, rather than a12-mer, and indeed some phage expressing 11-mers were isolated in theselections described above (see Table 2).

In the foregoing tables, Class I peptides only bind KDR in the absenceof heparin, and therefore presumably target the heparin binding domainof KDR; Class II peptides bind in the presence or absence of heparin orVEGF, and therefore presumably bind at a non-involved site on KDR; ClassIII peptides exhibit binding characteristics that are not affected byheparin but are perturbed in the presence of VEGF, and thereforepresumably these bind either to VEGF or the VEGF binding domain of KDR.NA signifies data not available. In the elution column, 1 HR, O/N, andCell stand for 1 hour VEGF, overnight VEGF, and bead infection elutions,respectively. In some cases, a particular isolate sequence was observedin two different elutions. For the isolates identified by secondgeneration library, VEGF elutions were substituted with peptide elutions(see below).

Example 2 Peptide Synthesis and Fluorescein Labeling

Selected KDR or VEGF/KDR complex binding peptides corresponding topositive phage isolates were synthesized on solid phase using9-fluorenylmethoxycarbonyl protocols and purified by reverse phasechromatography. Peptide masses were confirmed by electrospray massspectrometry, and peptides were quantified by absorbance at 280 nm. Forsynthesis, two N-terminal and two C-terminal amino acids from the phagevector sequence from which the peptide was excised were retained, and a-Gly-Gly-Gly-Lys-NH₂ linker (SEQ ID NO: 262) was added to the C-terminusof each peptide. Each peptide was N-terminally acetylated. For peptideswith selected lysine residues, these were protected with1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),which allows selective coupling to the C-terminal lysine, is not removedduring peptide cleavage, and can be removed after coupling with 2%hydrazine in DMF or 0.5 M hydroxylamine, pH 8, in water.

Each peptide was labeled with fluorescein on the C-terminal lysine usingfluorescein (N-hydroxysuccinimide ester derivative) or fluoresceinisothiocyanate (FITC) in DMF, 2% diisopropylethylamine (DIPEA). If thepeptide contained an ivDde protected lysine, the reaction was quenchedby the addition of 2% hydrazine, which reacts with all freeNHS-fluorescein and removes the internal protecting group. For all otherpeptides, the reaction was quenched by the addition of an equal volumeof 0.5 M hydroxylamine, pH 8. The quenched reactions were then dilutedwith water to less than 10% DMF and then purified using C18 reversephase chromatography. The peptides were characterized for purity andcorrect mass on an LC-MS system (HP1100 HPLC with in-line SCIEX AP150single quadrapole mass spectrometer).

Example 3 Fluorescence Anisotropy Measurements and BiaCore Assays

Fluorescence anisotropy measurements were performed in 384-wellmicroplates in a volume of 10 μl in binding buffer (PBS, 0.01% Tween-20,pH 7.5) using a Tecan Polarion fluorescence polarization plate reader.In some cases, heparin (0.5 μg/mL) or 10% human serum was added to thebinding buffer (data not shown). The concentration of fluoresceinlabeled peptide was held constant (20 nM) and the concentration ofKDR-Fc (or similar target) was varied. Binding mixtures wereequilibrated for 10 minutes in the microplate at 30° C. beforemeasurement. The observed change in anisotropy was fit to the equationbelow via nonlinear regression to obtain the apparent K_(D). Thisequation (1) assumes that the synthetic peptide and KDR form areversible complex in solution with 1:1 stoichiometry.

$\begin{matrix}{{r_{obs} = {r_{free} + {( {r_{bound} - r_{free}} )\frac{( {K_{D} + {KDR} + P} ) - \sqrt{( {K_{D} + {KDR} + P} )^{2} - {4 \cdot {KDR} \cdot P}}}{2 \cdot P}}}},} & (1)\end{matrix}$where r_(obs) is the observed anisotropy, r_(free) is the anisotropy ofthe free peptide, r_(bound) is the anisotropy of the bound peptide,K_(D) is the apparent dissociation constant, KDR is the total KDRconcentration, and P is the total fluorescein-labeled peptideconcentration. K_(D) was calculated in a direct binding assay (K_(D,B))(see Table 8), and therefore these values represent KDR binding to thefluorescein labeled peptide.

For BiaCore determinations of K_(D), KDR-Fc (or other protein targets)was cross-linked to the dextran surface of a CM5 sensor chip by thestandard amine coupling procedure (0.5 mg/mL solutions diluted 1:20 with50 mM acetate, pH 6.0, R_(L) KDR-Fc=12859). Experiments were performedin HBS-P buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% polysorbate20 (v/v)). Peptide solutions quantitated by extinction coefficient werediluted to 400 nM in HBS-P. Serial dilutions were performed to produce200, 100, 50, and 25 nM solutions. For association, peptides wereinjected at 20 μl/min. for 1 minute using the kinject program. Followinga 1-minute dissociation, any remaining peptide was stripped from thetarget surface with a quick injection of 1M NaCl for 25 sec. at 50μl/min. All samples were injected in duplicate. Between each peptideseries a buffer injection and a non-target binding peptide injectionserved as additional controls. Sensorgrams were analyzed using thesimultaneous k_(a)/k_(d) fitting program in the BIAevaluation software3.1. Apparent K_(D) by this method is set forth as BiaK_(D) in Table 8.Unlike the fluorescence anisotropy experiments above, the unlabeledpeptide was used for all testing using this assay and therefore, thesevalues represent KDR binding to the unlabeled peptide. Bindingaffinities determined for the synthesized polypeptides are set forth inTable 8, below. The putative disulfide-constrained cyclic peptidemoieties of the polypeptides are in bold.

TABLE 8 Binding Affinities for Synthesized Peptides SequenceK_(D,B )(μM) BiaK_(D )(μM) SEQ ID NO: TN8 AGDSWCSTEYTYCEMIGTGGGK >2 263AGPKWCEEDWYYCMITGTGGGK 0.28 0.027 264 AGVWECAKTFPFCHWFGTGGGK 2.60 265AGWVECWWKSGQCYEFGTGGGK 1.3 266 AGWLECYAEFGHCYNFGTGGGK >10 267AGWIQCNSITGHCTSGGTGGGK 0.24 268 AGWIECYHPDGICYHFGTGGGK 0.32 0.32 269AGSDWCRVDWYYCWLMGTGGGK 0.064 270 AGANWCEEDWYYCFITGTGGGK 0.310 271AGANWCEEDWYYCWITGTGGGK 0.097 272 AGPDWCEEDWYYCWITGTGGGK 0.075 273AGSNWCEEDWYYCYITGTGGGK 0.046 274 AGPDWCAADWYYCYITGTGGGK 0.057 275AGPEWCEVDWYYCWLLGTGGGK 0.075 276 AGPTWCEDDWYYCWLFGTGGGK 0.0032 0.079 277AGSKWCEQDWYYCWLLGTGGGK 0.400 278 AGRNWCEEDWYYCFITGTGGGK 0.190 279AGVNWCEEDWYYCWITGTGGGK 0.260 280 AGANWCEEDWYYCYITGTGGGK 0.180 281AGQAWVECYAETGYCWPRSWGTGGGK 0.71 282 AGQAWIECYAEDGYCWPRSWGTGGGK 1.40 283AGVGWVECYQSTGFCYHSRDGTGGGK 1.30 284 AGFTWVECHqATGRCVEWTTGTGGGK 2.00 285AGDWWVECRVGTGLCYRYDTGTGGGK 0.93 286 AGDSWVECDAQTGFCYSFLYGTGGGK 2.30 287AGGGWVECYWATGRCIEFAGGTGGGK NB 288 AGERWVECRAETGFCYTWVSGTGGGK 2.10 289AGGGWVECRAETGHCQEYRLGTGGGK 1.60 290 AGVAWVECYQTTGKCYTFRGGTGGGK ~2 291AGEGWVECFANTGACFTYPRGTGGGK 2.10 292 TN12 GDYPWCHELSDSVTRFCVPWDPGGGK 0.980.18 293 GDSRVCWEDSWGGEVCFRYDPGGGK 0.069 0.12 294GDDHMCRSPDYQDHVFCMYWDPGGGK 0.48 0.14 295 GDPPLCYFVGTQEWHHCNPFDPGGGK 0.60296 GDDSYCMMNEKGWWNCYLYDPGGGK 1.3 297 GDPAQCWESNYQGIFFCDNPDPGGGK 2.3 298GDGSWCEMRQDVGKWNCFSDDPGGGK 0.62 0.18 299 GDGWACAKWPWGGEICQPSDPGGGK 1.01.5 300 GDPDTCTMWGDSGRWYCFPADPGGGK 0.49 0.26 301GDNWKCEYTQGYDYTECVYLDPGGGK 0.82 302 GDNWECGWSNMFQKEFCARPDPGGGK 0.21 0.99303 GDWWECKREEYRNTTWCAWADPGGGK 486 GDSSVCFEYSWGGEVCFRYDPGGGK 0.058 487GDSRVCWEYSWGGQICLGYDPGGGK 0.32 488 Lin20 AQQVQYQFFLGTPRYEQWDLDKGGK 1.7304 AQEPEGYAYWEVITLYHEEDGDGGK 0.27 0.73 305 AQAFPRFGGDDYWIQQYLRYTDGGK0.53 0.25 306 AQGDYVYWEIIELTGATDHTPPGGK 0.18 307AQRGDYQEQYWHQQLVEQLKLLGGK 0.31 5.3 308 AQRSWYLGPPYYEEWDPIPNGGK 1.8 309AQDWYYDEILSMADQLRHAFLSGGGK 0.05 310 TN9 AGIDFCKGMAPWLCADMGTGGGK 0.730.18 311 AGPWTCWLEDHLACAMLGTGGGK 3.9 312 AGDWGCSLGNWYWCSTEGTGGGK 2.0 313TN10 GSDHHCYLHNGQWICYPFAPGGGK 0.26 0.15 314 GSNSHCYIWDGMWLCFPDAPGGGK0.74 315 MTN13 SGRLDCDKVFSGPYGKVCVSYGSGGGK 1.05 316SGRLDCDKVFSGPHGKICVNYGSGGGK ~2 317 SGRTTCHHQISGPHGKICVNYGSGGGK 0.65 318SGAHQCHHWTSGPYGEVCFNYGSGGGK ~2 319

For the analysis of those peptides that bind specifically to KDR/VEGFcomplex, each peptide was tested for binding to the complex in bothassays (fluorescence anisotropy/Biacore) as above. In the anisotropyassay, KDR-VEGF complex was formed by mixing together a two fold molarexcess of VEGF with KDR-Fc. This mixture was then used in the directbinding titration using a fluorescein labeled peptide as donepreviously. As a control, each peptide was also tested for binding toKDR and VEGF alone to assess their specificity for complex. Since noneof the peptides bound VEGF to any extent, the presence of excess VEGF inthe assay should not affect the K_(D) determination. As shown in Table9, below, all of the peptides showed a dramatic binding preference,binding for KDR/VEGF complex over VEGF. Some of them, however, did showsome residual binding to free KDR. To confirm the anisotropy results,the unlabeled peptides were tested in Biacore as before, except the chipwas saturated with VEGF to form KDR/VEGF complex prior to the injectionof the peptides. In the peptides tested, the BiaK_(D) was within atleast 2-fold of the anisotropy measurement.

TABLE 9 KDR/VEGF Complex Specific Peptides K_(D), B BiaK_(D) SEQ IDK_(D), B K_(D), B (KDR/ (KDR/ NO: Sequence (KDR) (VEGF) VEGF) VEGF) 320AGMPWCVEKDHWDCWWWGTGGGK NB 10 0.14 321 AGPGPCKGYMPHQCWYMGTGGGK 0.4 NB0.06 0.08 322 AGYGPCAEMSPWLCWYPGTGGGK 3.7 NB 0.13 323AGYGPCKNMPPWMCWHEGTGGGK 1.8 NB 0.18 0.42 324 AGGHPCKGMLPHTCWYEGTGGGK >10NB 3.3 325 AQAPAWTFGTNWRSIQRVDSLTGGGGGK NB NB 0.84 326AQEGWFRNPQEIMGFGDSWDKPGGGGGK NB NB 1.4

The putative disulfide-constrained cyclic peptide moiety is underscored.

Example 4 Preparation of KDR and VEGF/KDR Complex Binding Polypeptides

Utilizing the methods set forth above, biotinylated versions the KDR andVEGF/KDR complex binding polypeptides set forth in Table 10 wereprepared. The letter “J” in the peptide sequences refers to a spacer orlinker group, 8-amino-3,6-dioxaoctanoyl.

The ability of the biotinylated polypeptides (with the JJ spacer) tobind to KDR was assessed using the assay set forth in Example 5,following the procedures disclosed therein. Several biotinylatedpeptides bound well to the KDR-expressing cells: SEQ ID NO: 356 (K_(r))1.81 nM+/−0.27), SEQ ID NO: 264 (K_(D) 14.87+/−5.0 nM, four experimentaverage), SEQ ID NO: 294+spacer (K_(D) 10.00+/−2.36 nM, four experimentaverage), SEQ ID NO: 301 (K_(r)) 4.03+/−0.86 nM, three experimentaverage), SEQ ID NO: 337 (K_(D) 6.94+/−1.94 nM, one experiment), and SEQID NO: 338 (K_(D) 3.02+/−0.75 nM, one experiment).

TABLE 10 KDR, VEGF/KDR Complex Binding Polypeptides SEQ ID NO:Structure (or) Sequence Mol. Wt. MS 294 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK-NH₂2801.98 1399.6 [M − H]⁻ 329 Ac-AGMPWCVEKDHWDCWWGTGGGK-NH₂ 2730.14 — 311Ac-AGIDFCKGMAPWLCADMGTGGGK-NH₂ 2324.02 — 264Ac-AGPKWCEEDWYYCMITGTGGGK-NH₂ 2361 — 266 Ac-AGWVECWWKSGQCYEFGTGGGK-NH₂2474.06 — 330 Ac-AQEGWFRNPQEIMGFGDSWDKPGGGK-NH₂ 2934.35 — 299Ac-GDGSWCEMRQDVGK(iv-Dde)WNCFSDDP- 3075.29 1537.5 [M²⁻] GGGK-NH₂ 299Ac-GDGSWCEMRQDVGKWNCFSDDPGGGK-NH₂ 2869.16 — 303Ac-GDNWECGWSNMFQK(iv-Dde)EFCARPDP- 3160.36 1579.6 [M²⁻] GGGK-NH₂ 303Ac-GDNWECGWSNMFQKEFCARPDPGGGK-NH₂ 2954.23 — 294Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(Biotin)- 3030.29 1512.4 [M²⁻] NH₂ 331Ac-AQRGDYQEQYWHQQLVEQLK(iv-Dde)LLGGGK- 3318.71 1659.1 [M²⁻] NH₂ 331Ac-AQRGDYQEQYWHQQLVEQLKLLGGGK-NH₂ 3112.58 — 332Ac-AGWYWCDYYGIGCK(iv-Dde)WTGGGK-NH₂ 2673.18 333Ac-AGWYWCDYYGIGCKWTGTGGGK-NH₂ 2467.05 334Ac-AQWYYDWFHNQRKPPSDWIDNLGGGK-NH₂ 3218.51 — 323Ac-AGYGPCKNMPPWMCWHEGTGGGK-NH₂ 2502.05 — 335Ac-AGPKWCEEDWYYCMITGTGGGK(N,N-Dimethyl- 2836.204 2833.4 [M − H]⁻Gly-Ser-Cys(Acm)-Gly)-NH₂ 264 Ac-AGPK(iv-Dde)WCEEDWYYCMITGTGGGK-NH₂2698.11 2695.7 [M − H]⁻; 1347.8 [M − 2H]²⁻/2 336Ac-WQPCPWESWTFCWDPGGGK(AcSCH₂C(═O)—)—NH₂ 2422.71 2420.7 [M − H]⁻,1209.9 [M − 2H]/2 264 Ac-AGPKWCEEDWYYCMITGTGGGK(Biotin)-NH₂ 2718.132833.4 (M − H)⁻ 264 Ac-AGPKWCEEDWYYCMITGTGGGK(Biotin-JJ-)- 3008.441502.6.4 (M − 2H)²⁻/2 NH₂ 264 Ac-AGPKWCEEDWYYCMITGTGGGK 2608.961304, [M − 2H]²⁻/2 (AcSCH₂C(═O)—)—NH₂ 294Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(Biotin- 3316.4 1657.8, [M − 2H]²⁻/2JJ-)-NH₂ 294 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK 2917.15 1457.4, [M − 2H]²⁻/2(AcSCH₂C(═O)—)—NH₂ 294 Biotin-JJGDSRVCWEDSWGGEVCFRYDPGGGK- 3272.341636.7, [M − 2H]²⁻/2 NH₂ 264 Ac-AGPKWCEEDWYYCMITGT- 2899.28 1449.2, [M −2H]²⁻/2 GGGK(AcSCH₂C(═O)-JJ-)-NH₂ 277Ac-AGPTWCEDDWYYCWLFGTGGGK(Biotin-JJ-)- 3066.27 1532.8, [M − 2H]²⁻/2 NH₂337 Ac-VCWEDSWGGEVCFRYDPGGGK(Biotin-JJ-)-NH₂ 2903.24 1449.3, (M −2H)²⁻/2; 965.8, (M − 3H)³⁻/3 338 Ac-AGPTWCEDDWYYCWLFGTJK(Biotin-JJ-)-NH₂3042.44 1519.7, (M − 2H)²⁻/2−; 1012.8 (M − 3H)³⁻/3 294Ac-GDSRVCWEDSWGGEVCFRYDPGGGK 3208.48 1602.6, [M − 2H]²⁻/2(AcSCH₂C(═O)-JJ-)-NH₂ 339 Ac-AGPTWCEDDWYYCWLFGTGGGK(N,N- 3242.331621.5, [M − 2H]²⁻/2 Dimethyl-Gly-Ser-Cys(Acm)-Gly-JJ-)-NH₂ 277Ac-AGPTWCEDDWYYCWLFGTGGGK 2907.29 1453.1, [M − 2H]²⁻/2(AcSCH₂C(═O)-JJ-)-NH₂ 340 Ac-AQAHMPPWRPVAVDALFDWVEGG- 3404.641701.6, [M − 2H]²⁻/2 GGGK(Biotin-JJ-)-NH₂ 341Ac-AQAHMPPWWPLAVDAQEDWFEGG- 3493.59 1746.2, [M − 2H]²⁻/2GGGK(Biotin-JJ-)-NH₂ 342 Ac-AQAQMPPWWPLAVDALFDWFEGG- 3487.641743.2, [M − 2H]²⁻/2 GGGK(Biotin-JJ-)-NH₂ 343Ac-AQDWYWREWMPMHAQFLADDWGG- 3751.64 1874.3, [M − 2H]²⁻/2GGGK(Biotin-JJ-)-NH₂ 344 Ac-AQK(ivDde)K(iv-Dde)EDAQQWYWTDYVPSY- 4220.062108.9, [M − 2H]²⁻/2 LYRGGGGGK(Biotin-JJ-)-NH₂ 345Ac-AQPVTDWTPHHPK(iv-Dde)APDVWLFYT- 3781.86 1890.4, [M − 2H]²⁻/2GGGGGK(Biotin-JJ-)-NH₂ 346 Ac-AQDALEAPK(iv-Dde)RDWYYDWFLNHSP- 3897.851948.0, [M − 2H]²⁻/2 GGGGGK(Biotin-JJ-)-NH₂ 347Ac-KWCEEDWYYCMITGTGGGK(Biotin-JJ-)-NH₂ 2781.2 1390.0, [M − 2H]²⁻/2 348Ac-AGPKWCEEDWYYCMIGGGK(Biotin-JJ-)-NH₂ 2747.15 1373.5, [M − 2H]²⁻/2 349Ac-KWCEEDWYYCMIGGGK(Biotin-JJ-)-NH₂ 2522.04 1260.8, [M − 2H]²⁻/2 350Ac-AQPDNWK(iv-Dde)EFYESGWK(iv-Dde)- 4377.2 2188.4, [M − 2H]²⁻/2YPSLYK(iv-Dde)PLGGGGGK(Biotin-JJ-)-NH₂ 351 Ac-AQMPPGFSYWEQVVLHDDAQVLGG-3499.7 1749.2, [M − 2H]²⁻/2 GGGK(Biotin-JJ-)-NH₂ 352Ac-AQARMGDDWEEAPPHEWGWADGG- 3480.5 1740.2, [M − 2H]²⁻/2GGGK(Biotin-JJ-)-NH₂ 353 Ac-AQPEDSEAWYWLNYRPTMFHQLGG- 3751.71875.8, [M − 2H]²⁻/2 GGGK(Biotin-JJ-)-NH₂ 354Ac-AQSTNGDSFVYWEEVELVDHPGG- 3554.6 1776.4, [M − 2H]²⁻/2GGGK(Biotin-JJ-)-NH₂ 355 Ac-AQWESDYWDQMRQQLK(iv-Dde)TAYMK(iv- 4187.022093.0, [M − 2H]²⁻/2 Dde)VGGGGGK(Biotin-JJ-)-NH₂ 356 Ac- 3641.691820.9, [M − 2H]²⁻/2 AQDWYYDEILSMADQLRHAFLSGGGGGK(Biotin-JJ-)- NH₂ Theputative disulfide constrained cyclic peptide is indicated in bold.

Example 5 Binding of KDR Binding Peptides/Avidin HRP Complex to KDRTransfected 293H cells

To determine the binding of peptides identified by phage display to KDRexpressed in transiently-transfected 293H cells, a novel assay thatmeasures the binding of biotinylated peptides complexed with neutravidinHRP to KDR on the surface of the transfected cells was developed. Thisassay was used to screen the biotinylated peptides set forth in Example4. Neutravidin HRP was used instead of streptavidin or avidin because ithas lower non-specific binding to molecules other than biotin due to theabsence of lectin binding carbohydrate moieties and also due to theabsence of the cell adhesion receptor-binding RYD domain in neutravidin.

In the experiments described herein, tetrameric complexes of KDR-bindingpeptides SEQ ID NO: 294, SEQ ID NO: 264, SEQ ID NO: 277 and SEQ ID NO:356 and a control peptide, which does not bind to KDR, were prepared andtested for their ability to bind 293H cells that weretransiently-transfected with KDR. All four tetrameric complexes ofKDR-binding peptides were biotinylated and contained the JJ spacer, andbound to the KDR-expressing cells; however, SEQ ID NO: 356 exhibited thebest K_(D) (1.81 nM). The tetrameric complexes of KDR-binding peptidesSEQ ID NO: 294, SEQ ID NO: 264 exhibited improved binding over monomersof the same peptides. Moreover, inclusion of a spacer between theKDR-binding peptide and the biotin was shown to improve binding inExperiment B.

In Experiment C, it was shown that this assay can be used to assess theeffect of serum on binding of peptides of the invention to KDR andVEGF/KDR complex. The binding of SEQ ID NO: 264, SEQ ID NO: 294, and SEQID NO: 356 was not significantly affected by the presence of serum,while the binding of SEQ ID NO: 277 was reduced more than 50% in thepresence of serum.

In Experiment D, it was shown that this assay is useful in evaluatingdistinct combinations of KDR and VEGF/KDR complex binding polypeptidesfor use in multimeric targeting constructs that contain more than oneKDR and VEGF/KDR complex binding polypeptide. Moreover, Experiments Dand E establish that tetrameric constructs including two or more KDRbinding peptides that bind to different epitopes exhibited superiorbinding to “pure” tetrameric constructs of the targeting peptides alone.

Experiment A Preparation of m-RNA & 5′ RACE Ready cDNA Library

HUVEC cells were grown to almost 80% confluence in 175 cm² tissueculture flasks (Becton Dickinson, Biocoat, cat #6478) and then 10 ng/mLof bFGF (Oncogene, cat #PF003) was added for 24 h to induce expressionof KDR. mRNA was isolated using the micro-fast track 2.0 kit fromInvitrogen (cat. # K1520-02). 12 μg of mRNA (measured by absorbance at260 nM) was obtained from two flasks (about 30 million cells) followingthe kit instructions. Reverse transcription to generate cDNA wasperformed with 2 μg of mRNA, oligo dT primer (5′-(T)₂₅GC-3′) and/orsmart II oligo (5′AAGCAGTGGTAACAACGCAGAGTACGCGGG-3′) (SEQ ID NO: 357)using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. Thereaction was performed in a total volume of 20 μl and the reaction mixcontained 2 μl of RNA, 1 μl smart II oligo, 1 μl of oligo dT primer, 4μl of 5× first-strand buffer (250 mM Tris HCl pH 8.3, 375 mM KCl, 30 mMMgCl₂) 1 μl DTT (20 mM, also supplied with reverse transcriptase), 1 μldNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP in ddH₂O, Stratagene,cat. #200415), 9 μl ddH₂O and 1 μl MMLV reverse transcriptase(Clonetech, cat #8460-1). The reverse transcription reaction wasperformed for 90 minutes at 42° C., and the reaction was stopped byadding 250 μl of tricine-EDTA buffer (10 mM tricine, 1.0 mM EDTA). Thereverse transcription product, a 5′ RACE ready cDNA library, can bestored for 3 months at −20° C. Note that all water used for DNA and RNAapplication was DNAse and RNAse free from USB (cat. #70783).

Cloning of s-KDR into TOPOII Vector

In order to clone s-KDR, a 5′ oligo (G ATG GAG AGC AAG GTG CTG CTG G)(SEQ ID NO: 358) and a 3′ oligo (C CAA GTT CGT CTT TTC CTG GGC A) (SEQID NO: 359) were used. These were designed to amplify the completeextracellular domain of KDR (˜2.2 kbps) from the 5′ RACE ready cDNAlibrary (prepared above) using polymerase chain reaction (PCR) with pfupolymerase (Stratagene, cat. #600135). The PCR reaction was done intotal volume of 50 μl and the reaction mix contained 2 μl 5′ RACE readycDNA library, 1 μl 5′ oligo (10 μM), 1 μl 3′ oligo (10 μM), 5 μl 10×PCRbuffer [PCR buffer (200 mM Tris-HCl pH 8.8, 20 mM MgSO₄, 100 mM KCl, 100mM (NH₄)₂SO₄) supplied with pfu enzyme plus 1% DMSO and 8% glycerol], 1μl dNTP mix (10 mM) and 40 μl ddH₂0. The PCR reaction was performed byusing a program set for 40 cycles of 1 minute at 94 C, 1 minute at 68 Cand 4 minutes at 72 C. The PCR product was purified by extraction with 1volume of phenol, followed by extraction with 1 volume of chloroform andprecipitated using 3 volume of ethanol and 1/10 volume of 3M sodiumacetate. The PCR product was resuspended in 17 μl of ddH₂O, the 2 μl of10×Taq polymerase buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mMMgCl₂, 0.01% gelatin) and 1 μl of Taq polymerase (Stratagene, cat.#600131) was added to generate an A overhang to each end of the product.After incubating for 1 hour at 72 C the modified product was cloneddirectly into a TOPOII vector (InVitrogen, Carlsbad, Calif.) followingthe manufacturer's protocol to give TOPO-sKDR. The TOPO vector allowseasy cloning of PCR products because of the A-overhang in Taq (PCRenzyme)-treated PCR products.

Cloning the Transmembrane and Cytoplasmic Domains of KDR into Topo IIVector

In order to clone the transmembrane and cytoplasmic domains of KDR, a 5′oligo (TCC CCC GGG ATC ATT ATT CTA GTA GGC ACG GCG GTG) (SEQ ID NO: 360)and a 3′ oligo (C AGG AGG AGA GCT CAG TGT GGT C) (SEQ ID NO: 361) wereused. These were designed to amplify the complete transmembrane andcytoplasmic domains of KDR (˜1.8 kbps) from the 5′ RACE ready cDNAlibrary (described above) using polymerase chain reaction (PCR) with pfupolymerase. PCR reaction conditions and the program were exactly thesame as described above for s-KDR. Just as with the s-KDR sequence, thePCR product was purified using phenol chloroform extraction, treatedwith Taq polymerase and cloned into TOPOII vector from Invitrogen togive TOPO-CYTO.

Cloning of Full-Length KDR into pcDNA6 Vector

To create the full-length receptor, the extra-cellular domain and thecytoplasmic domain (with trans-membrane domain) were amplified by PCRseparately from TOPO-sKDR and TOPO-CYTO respectively and ligated laterto create the full-length receptor. An oligo with a Not1 site at the 5′end of the extracellular domain (A TAA GAA TGC GGC CGC AGG ATG GAG AGCAAG GTG CTG CTG G) (SEQ ID NO: 362) and an oligo complimentary to the 3′end of the extracellular domain (TTC CAA GTT CGT CTT TTC CTG GGC ACC)(SEQ ID NO: 363) were used to amplify by PCR the extracellular domainfrom TOPO-sKDR. Similarly, the 5′ oligo (ATC ATT ATT CTA GTA GGC ACG GCGGTG) (SEQ ID NO: 364) and the 3′ oligo, with a Not1 site (A TAA GAA TGCGGC CGC AAC AGG AGG AGA GCT CAG TGT GGT C) (SEQ ID NO: 365), were usedto amplify by PCR the cytoplasmic domain of KDR (with transmembranedomain) from TOPO-CYTO. Both PCR products were digested with Not1 andligated together to create the full-length receptor. The cDNA encodingthe full-length receptor was purified on an agarose gel and ligated intothe Not I site of the pcDNA6/V5-HisC vector. Purification of DNA andligation was done as described earlier for psKDR. The ligation reactionwas used to transform a culture of DH5α bacteria and a number ofindividual clones were analyzed for the presence and orientation ofinsert by restriction analysis of purified plasmid from each clone withEcoRI enzyme.

Cell Culture

293H cells were obtained from Invitrogen (cat. #11631) and grown asmonolayer culture in their recommended media plus 1 mL/L pen/strep(Invitrogen, cat. #15140-148). All the cells were grown in presence ofantibiotic for everyday culture but were split into antibiotic freemedia for 16-20 hours prior to transfection.

Preparation of DNA for Transfection

E. coli bacteria DH5α containing pf-KDR was streaked onto LB with 50μg/mL ampicillin (LB agar from US biologicals, cat. #75851 andampicillin from Sigma, cat. #A2804) plates from a glycerol stock andplates were left in a 37° C. incubator to grow overnight. Next morning,a single colony was picked from the plate and grown in 3 mL ofLB/ampicillin media (LB from US biologicals, cat. # US75852) at 37° C.After 8 hours, 100 μl of bacterial culture from the 3 mL tube wastransferred to 250 mL of LB/ampicillin media for overnight incubation at37° C. Bacteria were grown up with circular agitation in a 500 mL bottle(Beckman, cat. #355605) at 220 rpm in a Lab-Line incubator shaker. Thenext day, the bacterial culture was processed using maxi-prep kit(QIAGEN, cat. #12163). Generally, about 1 mg of plasmid DNA (asquantitated by absorbance at 260 nm) was obtained from 250 mL ofbacterial culture.

Transfection of 293H Cells in 96 Well Plate

Transfection was done as recommended in the lipofectamine 2000 protocol(Invitrogen, cat#11668-019) using a poly-D-lysine-coated 96 well plate.320 ng of KDR DNA (pc-DNA6-fKDR)/per well in 0.1 mL was used for 96 welltransfection. Transfection was done in serum-containing media, thetransfection reagent mix was removed from cells after 6-8 hours andreplaced with regular serum-containing medium. Transfection was done inblack/clear 96-well plates (Becton Dickinson, cat. #354640). The lefthalf of the plate (48 wells) were mock-transfected (with no DNA) and theright half of the plate was transfected with KDR cDNA. The cells were80-90% confluent at the time of transfection and completely confluentnext day, at the time of the assay, otherwise the assay was aborted.

Preparation of M199 Media

In order to prepare M199 media for the assay, one M199 medium packet(GIBCO, cat. #31100-035), 20 mL of 1 mM HEPES (GIBCO, cat. #15630-080)and 2 gm of DIFCO Gelatin (DIFCO, cat. #0143-15-1) were added to 950 mLof ddH₂O and the pH of the solution was adjusted to 7.4 by addingapproximately 4 mL of 1N NaOH. After pH adjustment, the M199 media waswarmed to 37° C. in a water bath for 2 hours to dissolve the gelatin,then filter sterilized using 0.2 μm filters (Corning, cat. #43109), andstored at 4° C. to be used later in the assay.

Preparation of Softlink Soft Release Avidin-Sepharose

SoftLink soft release avidin-sepharose was prepared by centrifuging thesepharose obtained from Promega (cat. # V2011) at 12,000 rpm for 2minutes, washing twice with ice cold water (centrifuging in-between thewashes) and resuspending the pellet in ice cold water to make a 50%slurry in ddH₂O. A fresh 50% slurry of avidin-sepharose was prepared foreach experiment.

Preparation of Peptide/Neutravidin HRP Solution

Biotinylated peptides SEQ ID NOS: 294, 264, 277, 356, and thenon-binding biotinylated control peptide were used to prepare 250 μMstock solutions in 50% DMSO and a 33 μM stock solution ofneutravidin-HRP was prepared by dissolving 2 mg of neutravidin-HRP(Pierce, cat. #31001) in 1 mL of ddH₂O (all polypeptides contained theJJ spacer). Peptide stock solutions were stored at −20° C., whereas theNeutravidin HRP stock solution was stored at −80° C. To preparepeptide/neutravidin-HRP complexes, 10 μl of 250 μM biotinylated peptidestock solution and 10 μl of 33 μM neutravidin-HRP were added to 1 mL ofM199 medium. This mixture was incubated on a rotator at 4° C. for 60minutes, followed by addition of 50 μl of soft release avidin-sepharose(50% slurry in ddH₂0) to remove excess peptides and another incubationfor 30 minutes on a rotator at 4° C. Finally, the soft releaseavidin-sepharose was pelleted by centrifuging at 12,000 rpm for 5minutes at room temperature, and the resulting supernatant was used forthe assays. Fresh peptide/neutravidin-HRP complexes were prepared foreach experiment.

Preparation of Peptide/Neutravidin HRP Dilutions for the Assay

For saturation binding experiments, 120 μl, 60 μl, 20 μl, 10 μl, 8 μl, 6μl, 4 μl, and 1 μl of peptide/neutravidin HRP complex were added to 1.2mL aliquots of M199 medium to create dilutions with final concentrationsof 33.33 nM, 16.65 nM, 5.55 nM, 2.78 nM, 1.67 nM, 1.11 nM and 0.28 nMcomplex, respectively.

Preparation of Blocking Solution for Transfected 293H Cells

Blocking solution was prepared by adding 20 mL of M199 medium to 10 mgof lyophilized unlabeled neutravidin (Pierce, cat. #31000). Freshblocking solution was used for each experiment.

Assay to Detect the Binding of Peptide/Neutravidin-HRP

24 hours after transfection, each well of the 293H cells was washed oncewith 100 μl of M199 medium and incubated with 80 μl of blocking solutionat 37° C. After one hour, cells were washed twice with 100 μl of M199media and incubated with 70 μl of peptide/neutravidin-HRP dilutions ofcontrol peptide, SEQ ID NO: 264, SEQ ID NO: 294, SEQ ID NO: 277, and SEQID NO: 356 for two and half hours at room temperature. Each dilution wasadded to three separate wells of mock as well as KDR-transfected 293Hcells (two plates were used for each saturation binding experiment).After incubation at room temperature, plates were transferred to 4° C.for another half-hour incubation. Subsequently, cells were washed 5times with ice-cold M199 media and once with ice-cold PBS (in thatorder). After the final wash, 100 μl of ice cold TMB solution (KPL, cat.#50-76-00) was added to each well and each plate was incubated for 30minutes at 37° C. in an air incubator. Finally, the HRP enzyme reactionwas stopped by adding 50 μl of 1N phosphoric acid to each well, andbinding was quantitated by measuring absorbance at 450 nm using amicroplate reader (BioRad Model 3550).

Binding of Peptide/Neutravidin HRP to KDR-Transfected Cells

In this assay, complexes of control peptide, SEQ ID NO: 264, SEQ ID NO:294, SEQ ID NO: 277, and SEQ ID NO: 356 peptides, each biotinylated withthe JJ spacer and conjugated with neutravidin-HRP, were prepared asdescribed above and tested for their ability to bind 293H cells thatwere transiently-transfected with KDR. During the peptide/neutravidincomplex preparation, a 7.5-fold excess of biotinylated peptides overneutravidin-HRP was used to make sure that all four biotin binding siteson neutravidin were occupied. After complex formation, the excess offree biotinylated peptides was removed using soft releaseavidin-sepharose to avoid any competition between free biotinylatedpeptides and neutravidin HRP-complexed biotinylated peptides. Theexperiment was performed at several different concentrations ofpeptide/neutravidin-HRP, from 0.28 nM to 33.33 nM, to generatesaturation binding curves for SEQ ID NO: 264 and SEQ ID NO: 294 (FIG.1A) and 0.28 to 5.55 nM to generate saturation binding curve for SEQ IDNO: 277 and SEQ ID NO: 356 (FIG. 1B). In order to draw the saturationbinding curve, the background binding to mock-transfected cells wassubtracted from the binding to KDR-transfected cells for each distinctpeptide/neutravidin HRP complex at each concentration tested. Therefore,absorbance on the Y-axis of FIG. 1 is differential absorbance (KDR minusmock) and not the absolute absorbance. Analysis of the saturationbinding data in FIG. 1 using Graph Pad Prism software (version 3.0)yielded a K_(D) of 10.00 nM (+/−2.36) for the tetrameric SEQ ID NO: 294,14.87 nM (+/−5.066) for the tetrameric SEQ ID NO: 264, 4.031 nM(+/−0.86) for the tetrameric SEQ ID NO: 277, and 1.814 nM (+/−0.27) forthe tetrameric SEQ ID NO: 356 peptide complexes. These binding constantsare, as expected, lower than those measured by FP against the KDRFcconstruct for the related monodentate peptides SEQ ID NO: 294 (69 nM),SEQ ID NO: 264 (280 nM), SEQ ID NO: 310 (51 nM), but similar tomonodentate peptide SEQ ID NO: 277 (3 nM). As expected, no saturation ofbinding for the control (non-binder) peptide/neutravidin HRP-complex wasobserved. The binding of peptide/neutravidin HRP complexes (FIG. 2) at asingle concentration (5.55 nM) was plotted to demonstrate that a singleconcentration experiment can be used to differentiate between a KDRbinding peptide (SEQ ID NOS: 264, 294 and 277) from a non-bindingpeptide.

Experiment B

Experiment B was designed to look at the requirement of spacer (JJ,Table 10) between the KDR binding sequences (SEQ ID NOS: 294 and 264)and biotin. In this experiment, biotinylated peptides with and withoutspacer JJ were tested (e.g., biotinylated SEQ ID NO: 264 with the JJspacer, biotinylated SEQ ID NO: 264 without the JJ spacer, SEQ ID NO:294 with a spacer, and biotinylated SEQ ID NO: 294 without the spacer),and a non-KDR binding, biotinylated control peptide (with and withoutspacer, prepared as set forth above) was used as a control. The peptidestructure of all the KDR-binding sequences tested in this experiment isshown in FIG. 3.

This experiment was performed as set forth in Experiment A describedabove, except that it was only done at a single concentration of 2.78nM.

Results: It is evident from the results shown in the FIG. 4 that thespacer enhances binding of SEQ ID NO: 294 and SEQ ID NO: 264. The spacerbetween the binding sequence and biotin can be helpful in enhancingbinding to target molecule by multiple mechanisms. First, it could helpreduce the steric hindrance between four biotinylated peptides aftertheir binding to a single avidin molecule. Second, it could provideextra length necessary to reach multiple binding sites available on asingle cell.

Experiment C

Experiment C was designed to look at the serum effect on the binding ofSEQ ID NOS: 294, 264, 277 and 356. In this procedure, biotinylatedpeptide/avidin HRP complexes of SEQ ID NOS: 294, 264, 277 and 356 weretested in M199 media (as described above in Experiment A) with andwithout 40% rat serum. This experiment was performed as described forExperiment A except that it was only done at single concentration of6.66 nM for SEQ ID NOS: 294 and 264, 3.33 nM for SEQ ID NO: 277 and 2.22nM for SEQ ID NO: 356. Each of the polypeptides were biotinylated andhad the JJ spacer.

Results: Results in FIG. 5 indicate that binding of SEQ ID NO: 264, SEQID NO: 294, and SEQ ID NO: 356 was not significantly affected by 40% ratserum, whereas binding of SEQ ID NO: 277 (c) was more than 50% lower inpresence of 40% rat serum. More than an 80% drop in the binding ofTc-labeled SEQ ID NO: 277 with Tc-chelate was observed in the presenceof 40% rat serum (FIG. 97). Since the serum effect on the binding ofTc-labeled SEQ ID NO: 277 is mimicked in the avidin HRP assay disclosedherein, this assay may be used to rapidly evaluate the serum effect onthe binding of peptide(s) to KDR.

Experiment D

Experiment D was designed to evaluate the binding of tetramericcomplexes of KDR and VEGF/KDR complex binding polypeptides SEQ ID NO:294 and SEQ ID NO: 264, particularly where the constructs included atleast two KDR binding polypeptides. The KDR binding peptides and controlbinding peptide were prepared as described above. This experiment wasperformed using the protocol set forth for Experiment A, except theprocedures set forth below were unique to this experiment.

Preparation of Peptide/Neutravidin Solutions:

250 μM stock solutions of biotinylated peptides SEQ ID NOs: 264, 294 andcontrol peptide were prepared in 50% DMSO and a 33 μM stock solution ofNeutravidin HRP was prepared by dissolving 2 mg of Neutravidin HRP(Pierce, cat. #31001) in 1 mL of ddH₂O. Peptide stock solutions werestored at −20 C, whereas the Neutravidin HRP stock solution was storedat −80 C. The sequences of the biotinylated peptides are shown above. Toprepare peptide/neutravidin HRP complexes, a total 5.36 μL, of 250 μMbiotinylated peptide stock solution (or a mixture of peptide solutions,to give peptide molecules four times the number of avidin HRP molecules)and 10 μL, of 33 μM Neutravidin HRP were added to 1 mL of M199 medium.This mixture was incubated on a rotator at 4 C for 60 minutes, followedby addition of 50 μL, of soft release avidin-sepharose (50% slurry inddH₂0) to remove excess peptides and another incubation for 30 minuteson a rotator at 4 C. Finally, the soft release avidin-sepharose waspelleted by centrifuging at 12,000 rpm for 5 minutes at roomtemperature, and the resulting supernatant was used for the assays.Fresh peptide/neutravidin HRP complexes were prepared for eachexperiment.

Assay to Detect the Binding of Peptide/Neutravidin HRP:

24 hours after transfection, each well of the 293H cells was washed oncewith 100 μL, of M199 medium and incubated with 80 μL, of blockingsolution at 37 C. After one hour, cells were washed twice with 100 μL,of M199 media and incubated with 70 μL, of 3.33 nM peptide (or peptidemix)/neutravidin HRP solutions (prepared by adding 10 μL, of stockprepared earlier to 1 mL of M199 media) for two and half hours at roomtemperature. Each dilution was added to three separate wells of mock aswell as KDR-transfected 293H cells. After incubation at roomtemperature, plates were transferred to 4 C for another half-hourincubation. Subsequently, cells were washed five times with ice-coldM199 media and once with ice-cold PBS (in that order). After the finalwash, 100 μL, of ice cold TMB solution (KPL, Gaithersburg, Md.) wasadded to each well and each plate was incubated for 30 minutes at 37 Cin an air incubator. Finally, the HRP enzyme reaction was stopped byadding 50 μL, of 1N phosphoric acid to each well, and binding wasquantitated by measuring absorbance at 450 nm using a microplate reader(BioRad Model 3550).

Results:

This experiment establishes that SEQ ID NO: 294 and SEQ ID NO: 264 bindto KDR in multimeric fashion, and cooperate with each other for bindingto KDR in 293H transfected cells. A biotinylated control peptide thatdoes not bind to KDR was used. As expected, a tetrameric complex of thecontrol peptide with avidin-HRP did not show enhanced binding toKDR-transfected cells. Tetrameric complexes of SEQ ID NO: 294 and SEQ IDNO: 264 bound to KDR-transfected cells significantly better than tomock-transfected cells (see FIG. 6). SEQ ID NO: 294 tetramers, however,bound much better than SEQ ID NO: 264 tetramers. If the control peptidewas added to the peptide mixture used to form the tetrameric complexes,the binding to the KDR-transfected cells decreased. The ratio ofspecific binding of tetramer to monomer, dimer and trimer was calculatedby dividing the specific binding (obtained by subtracting the binding tomock transfected cells from KDR transfected cells) of tetramer, trimerand dimer with that of monomer. Results indicate that there isco-operative effect of multimerization of SEQ ID NOS: 264, 294 and 356on the binding to KDR-transfected cells.

Tetramer Trimer Dimer SEQ ID NO: 264 45.4 5 4.3 SEQ ID NO: 294* 38.6 7.12.7 SEQ ID NO: 277 1 1.1 1.1 SEQ ID NO: 356 16 5.7 2.3 *monomericpeptide binding at 2.22 nM was zero, therefore ratios were calculatedusing binding at 5.55 nM.

A mixture of 25% non-binding control peptide with 75% SEQ ID NO: 264 didnot bind significantly over background to KDR-transfected cells,indicating that multivalent binding is critical for the SEQ ID NO:264/avidn-HRP complex to remain bound to KDR throughout the assay. Thisphenomenon also held true for SEQ ID NO: 294, where substituting 50% ofthe peptide with control peptide in the tetrameric complex abolishedalmost all binding to KDR on the transfected cells.

Surprisingly, a peptide mixture composed of 50% control peptide with 25%SEQ ID NO: 294 and 25% SEQ ID NO: 264 bound quite well toKDR-transfected cells relative to mock-transfected cells, indicatingthat there is a great advantage to targeting two sites or epitopes onthe same target molecule. Furthermore, it was noted that tetramericcomplexes containing different ratios of SEQ ID NO: 294 and SEQ ID NO:264 (3:1, 2:2, and 1:3) all bound much better to KDR-transfected cellsthan pure tetramers of either peptide, in agreement with the idea thattargeting two distinct sites on a single target molecule is superior tomultimeric binding to a single site. This may be because multimericbinding to a single target requires that the multimeric binding entityspan two or more separate target molecules that are close enoughtogether for it to bind them simultaneously, whereas a multimeric binderthat can bind two or more distinct sites on a single target moleculedoes not depend on finding another target molecule within its reach toachieve multimeric binding.

Experiment E

Experiment E was designed to confirm that SEQ ID NO: 294 and SEQ ID NO:264 bind to distinct sites (epitopes) on KDR. If these peptides bind tothe same site on KDR, then they should be able to compete with eachother; however, if they bind to different sites they should not compete.This experiment was performed using a single concentration of SEQ ID NO:264/avidin HRP (3.33 nM) solution in each well and adding a varyingconcentration (0-2.5 μM) of biotinylated control peptide with spacer,SEQ ID NO: 264 and SEQ ID NO: 294, none of which were complexed withavidin.

Results:

It is evident from FIG. 7 that SEQ ID NO: 264 does compete with SEQ IDNO: 264/avidin HRP solution for binding to recombinant KDR-Fc fusionprotein whereas control peptide and SEQ ID NO: 294 do not compete withSEQ ID NO: 264/avidin HRP solution for binding to recombinant KDR-Fcfusion protein. Thus, SEQ ID NO: 264 and SEQ ID NO: 294 bind to distinctand complementary sites on KDR.

Example 6 Binding of Analogs of a KDR-Binding Peptide to KDR-ExpressingCells

N-terminal and C-terminal truncations of a KDR binding polypeptide weremade and the truncated polypeptides tested for binding to KDR-expressingcells. The synthesized polypeptides are shown in FIG. 8. Binding of thepolypeptides to KDR-expressing cells was determined following theprocedures of Example 3.

All of the peptides were N-terminally acetylated and fluoresceinated fordetermining apparent K_(D) according to the method described above(Example 3). The results indicate that, for the SEQ ID NO: 294 (FIG. 8)polypeptide, the C-terminal residues outside the disulfide-constrainedloop contribute to KDR binding.

Example 7 Bead-binding Assay to Confirm Ability of Peptides Identifiedby Phage Display to Bind KDR-Expressing Cells

The following procedures were performed to assess the ability ofKDR-binding peptides to bind to KDR-expressing cells. In this procedure,KDR-binding peptides containing SEQ ID NOS: 264, 337, 363, and 373 wereconjugated to fluorescent beads, and their ability to bind toKDR-expressing 293H cells was assessed. The experiments show thesepeptides can be used to bind particles such as beads to KDR-expressingsites. The results indicate that the binding of both KDR bindingsequences improved with the addition of a spacer.

Protocol

Biotinylation of an Anti-KDR Antibody:

Anti-KDR from Sigma (V-9134), as ascites fluid, was biotinylated using akit from Molecular Probes (F-6347) according to the manufacturer'sinstructions.

Preparation of Peptide-Conjugated Fluorescent Beads:

0.1 mL of a 0.2 mM stock solution of each biotinylated peptide (preparedas set forth above, in 50% DMSO) was incubated with 0.1 mL ofNeutravidin-coated red fluorescent microspheres (2 micron diameter,custom-ordered from Molecular Probes) and 0.2 mL of 50 mM MES (SigmaM-8250) buffer, pH 6.0 for 1 hour at room temperature on a rotator. As apositive control, biotinylated anti-KDR antibody was incubated with theNeutravidin-coated beads as above, except that 0.03 mg of thebiotinylated antibody preparation in PBS (Gibco #14190-136) was usedinstead of peptide solution. Beads can be stored at 4° C. until neededfor up to 1 week.

Binding Assay:

From the above bead preparations, 0.12 mL was spun for 10 minutes at2000 rpm in a microcentrifuge at room temperature. The supernatant wasremoved and 0.06 mL of MES pH 6.0 was added. Each bead solution was thenvortexed and sonicated in a water bath 15 min. To 1.47 mL of DMEM, highglucose (GIBCO #11965-084) with 1×MEM Non-Essential Amino Acids Solution(NEAA) (GIBCO 11140-050) and 40% FBS (Hyclone SH30070.02) 0.03 mL of thesonicated bead preparations was added. 96-well plates seeded with 293Hcells that have been mock-transfected in columns 1 to 6, andKDR-transfected in columns 7 to 12 (as in Example 5), were drained andwashed once with DMEM, high glucose with 1×NEAA and 40% FBS. To eachwell 0.1 mL of bead solution was added, six wells per bead preparation.After incubating at room temperature for 30 minutes, the wells weredrained by inverting the plates and washed four times with 0.1 mL PBSwith Ca⁺⁺Mg⁺⁺ (GIBCO #14040-117) with shaking at room temperature for 5minutes each wash. After draining, 0.1 mL of PBS was added per well. Theplates were then read on a Packard FluoroCount fluorometer at excitation550 nm/emission 620 nm. Unconjugated neutravidin beads were used as anegative control while beads conjugated with a biotinylated anti-KDRantibody were used as the positive control for the assay.

To calculate the number of beads bound per well, a standard curve withincreasing numbers of the same fluorescent beads was included in eachassay plate. The standard curve was used to calculate the number ofbeads bound per well based on the fluorescence intensity of each well.

Results:

The positive control beads with anti-KDR attached clearly boundpreferentially to the KDR-expressing cells while avidin beads withnothing attached did not bind to either cell type (FIG. 9). BiotinylatedSEQ ID NO: 264 beads did not bind to the KDR-transfected cellssignificantly more than to mock-transfected cells, but adding ahydrophilic spacer between the peptide moiety and the biotin group(biotinylated SEQ ID NO: 264 with a JJ spacer beads) enhanced binding toKDR cells without increasing the binding to mock-transfected cells.Biotinylated SEQ ID NO: 294 beads showed greater binding toKDR-transfected cells, and adding a hydrophilic spacer between thepeptide portion and the biotin of the molecule (biotinylated SEQ ID NO:294 with the JJ spacer) significantly improved the specific binding toKDR in the transfected cells. Thus, the peptide sequences of both SEQ IDNO: 264 and SEQ ID NO: 294 can be used to bind particles such as beadsto KDR-expressing sites. Addition of a hydrophilic spacer between thepeptide and the group used for attachment to the particle shouldroutinely be tested with new targeting molecules as it improved thebinding for both of the peptides evaluated here.

Example 8 Competition of KDR Binding Peptides and ¹²⁵I Labeled VEGF forBinding to KDR-Transfected 293H Cells

KDR-binding polypeptides were next assessed for their ability to competewith 125I-labeled VEGF for binding to KDR expressed by transfected 293Hcells. The results indicate that KDR-binding polypeptide SEQ ID NO: 263(Ac-AGDSWCSTEYTYCEMIGTGGGK-NH₂) did not compete significantly with¹²⁵I-labeled VEGF, and SEQ ID NOS: 294, 264, and SEQ ID NO: 277 competedvery well with ¹²⁵I-labeled VEGF, inhibiting 96.29+2.97% and104.48+2.074% of ¹²⁵I-labeled VEGF binding.

Transfection of 293H Cells:

293H cells were transfected using the protocol described in Example 5.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. #354640). The left half of the plates (48 wells) weremock-transfected (with no DNA) and the right half of the plates weretransfected with KDR cDNA. The cells were 80-90% confluent at the timeof transfection and completely confluent the next day, at the time ofthe assay; otherwise the assay was aborted.

Preparation of M199 Media:

M199 medium was prepared as described in Example 5.

Preparation of Peptide Solutions:

3 mM stock solutions of peptides SEQ ID NO: 294, SEQ ID NO: 263, SEQ IDNO: 264 and SEQ ID NO: 277 were prepared as described above in 50% DMSO.

Preparation of ¹²⁵I-Labeled VEGF Solution for the Assay:

25 μCi of lyophilized ¹²⁵I-labeled VEGF (Amersham, cat. # IM274) wasreconstituted with 250 μl of ddH₂O to create a stock solution, which wasstored at −80 C for later use. For each assay, a 300 μM solution of¹²⁵I-labeled VEGF was made fresh by diluting the above stock solution inM199 medium. The concentration of ¹²⁵I-labeled VEGF was calculated dailybased on the specific activity of the material on that day.

Preparation of 30 μM and 0.3 μM Peptide Solution in 300 pM ¹²⁵ I-labeledVEGF:

For each 96 well plate, 10 mL of 300 pM ¹²⁵I-labeled VEGF in M199 mediumwas prepared at 4° C. Each peptide solution (3 mM, prepared as describedabove) was diluted 1:100 and 1:10000 in 300 μl of M199 media with 300 pM¹²⁵I-labeled VEGF to prepare 30 μM and 0.3 μM peptide solutionscontaining 300 pM of ¹²⁵I-labeled VEGF. Once prepared, the solutionswere kept on ice until ready to use. The dilution of peptides in M199media containing 300 pM ¹²⁵I-labeled VEGF was done freshly for eachexperiment.

Assay to Detect Competition with ¹²⁵ I-Labeled VEGF in 293H Cells:

Cells were used 24 hours after transfection, and to prepare the cellsfor the assay, they were washed 3 times with room temperature M199medium and placed in the refrigerator. After 15 minutes, the M199 mediumwas removed from the plate and replaced with 75 μl of 300 pM¹²⁵I-labeled VEGF in M199 medium (prepared as above) with thepolypeptides. Each dilution was added to three separate wells of mockand KDR transfected cells. After incubating at 4° C. for 2 hours, theplates were washed 5 times with cold binding buffer, gently blotted dryand checked under a microscope for cell loss. 100 μl of solubilizingsolution (2% Triton X-100, 10% Glycerol, 0.1% BSA) was added to eachwell and the plates were incubated at room temperature for 30 minutes.The solubilizing solution in each well was mixed by pipeting up anddown, and transferred to 1.2 mL tubes. Each well was washed twice with100 μl of solubilizing solution and the washes were added to thecorresponding 1.2 mL tube. Each 1.2 mL tube was then transferred to a15.7×100 cm tube to be counted in an LKB Gamma Counter using program 54(¹²⁵I window for 1 minute).

Competition of Peptides with ¹²⁵I-Labeled VEGF in 293H Cells:

The ability of KDR-binding peptides SEQ ID NO: 294, SEQ ID NO: 263, SEQID NO: 264 and SEQ ID NO: 277 to specifically block ¹²⁵I-labeled VEGFbinding to KDR was assessed in mock-transfected and KDR-transfectedcells. SEQ ID NO: 263 was used in the assay as a negative control, as itexhibited poor binding to KDR in the FP assays described herein andwould therefore not be expected to displace or compete with VEGF. Tocalculate the specific binding to KDR, the binding of ¹²⁵I-labeled VEGFto mock-transfected cells was subtracted from KDR-transfected cells.Therefore, the binding of ¹²⁵I-labeled VEGF to sites other than KDR(which may or may not be present in 293H cells) is not included whencalculating the inhibition of ¹²⁵I-labeled VEGF binding to 293H cells byKDR-binding peptides. Percentage inhibition was calculated using formula[(Y1−Y2)*100/Y1], where Y1 is specific binding to KDR-transfected 293Hcells in the absence peptides, and Y2 is specific binding toKDR-transfected 293H cells in the presence of peptides or DMSO. Specificbinding to KDR-transfected 293H cells was calculated by subtractingbinding to mock-transfected 293H cells from binding to KDR-transfected293H cells.

As shown in FIG. 10, in 293 cells, SEQ ID NO: 263, which due to itsrelatively high K_(d) (>2 μM) was used as a negative control, did notcompete significantly with ¹²⁵I-labeled VEGF, 12.69±7.18% at 30 μM and−5.45±9.37% at 0.3 μM (FIG. 10). At the same time, SEQ ID NOS: 294 and277 competed very well with ¹²⁵I-labeled VEGF, inhibiting 96.29±2.97%and 104.48±2.074% of ¹²⁵I-labeled VEGF binding at 30 μM and 52.27±3.78%and 80.96±3.8% at 0.3 μM (FIG. 10) respectively. The percentageinhibition with SEQ ID NO: 264 was 47.95±5.09% of ¹²⁵I-labeled VEGFbinding at 30 μM and 24.41±8.43% at 0.3 μM (FIG. 10). Thus, the threestrongly KDR-binding polypeptides did compete with VEGF, and theirpotency increased with their binding affinity. This assay will be usefulfor identifying peptides that bind tightly to KDR but do not competewith VEGF, a feature that may be useful for imaging KDR in tumors, wherethere is frequently a high local concentration of VEGF that wouldotherwise block the binding of KDR-targeting molecules.

Example 9 Inhibition of VEGF-Induced KDR Receptor Activation by PeptidesIdentified by Phage Display

The ability of KDR-binding peptides identified by phage display toinhibit VEGF induced activation (phosphorylation) of KDR was assessedusing the following assay. A number of peptides of the invention wereshown to inhibit activation of KDR in monomeric and/or tetramericconstructs. As discussed supra, peptides that inhibit activation of KDRmay be useful as anti-angiogenic agents.

Protocol

Human umbilical vein endothelial cells (HUVECs) (Biowhittaker Catalog#CC-2519) were obtained frozen on dry ice and stored in liquid nitrogenuntil thawing. These cells were thawed, passaged, and maintained asdescribed by the manufacturer in EGM-MV medium (Biowhittaker Catalog#CC-3125). Cells seeded into 100 mm dishes were allowed to becomeconfluent, then cultured overnight in basal EBM medium lacking serum(Biowhittaker Catalog #CC-3121). The next morning, the medium in thedishes was replaced with 10 mL fresh EBM medium at 37 C containingeither no additives (negative control), 5 ng/mL VEGF (Calbiochem Catalog#676472 or Peprotech Catalog #100-20) (positive control), or 5 ng/mLVEGF plus the indicated concentration of the KDR-binding peptide(prepared as described above). In some cases, a neutralizing anti-KDRantibody (Catalog #AF357, R&D Systems) was used as a positive controlinhibitor of activation. In such cases, the antibody was pre-incubatedwith the test cells for 30 min at 37° C. prior to the addition of freshmedium containing both VEGF and the antibody. After incubating thedishes 5 min. in a 37° C. tissue culture incubator they were washedthree times with ice-cold D-PBS containing calcium and magnesium andplaced on ice without removing the last 10 mL of Delbecco's phosphatebuffered saline (D-PBS). The first dish of a set was drained and 0.5 mLof Triton lysis buffer was added (20 mM Tris base pH 8.0, 137 mM NaCl,10% glycerol, 1% Triton X-100, 2 mM EDTA (ethylenediaminetetraaceticacid), 1 mM PMSF(phenylmethylsulfonylfluoride), 1 mM sodiumorthovanadate, 100 mM NaF, 50 mM sodium pyrophosphate, 10 μg/mLleupeptin, 10 μg/mL aprotinin) The cells were quickly scraped into thelysis buffer using a cell scraper (Falcon, Cat No. #353087), dispersedby pipeting up and down briefly, and the resulting lysate wastransferred to the second drained dish of the pair. Another 0.5 mL oflysis buffer was used to rinse out the first dish then transferred tothe second dish, which was then also scraped and dispersed. The pooledlysate from the two dishes was transferred to a 1.5 mL Eppindorf tube.The above procedure was repeated for each of the controls and testsamples (KDR-binding peptides), one at a time. The lysates were storedon ice until all the samples had been processed. At this point sampleswere either stored at −70° C. or processed to the end of the assaywithout interruption.

The lysates, freshly prepared or frozen and thawed, were precleared byadding 20 μl of protein A-sepharose beads (Sigma 3391, preswollen inD-PBS, washed three times with a large excess of D-PBS, andreconstituted with 6 mL D-PBS to generate a 50% slurry) and rocking at4° C. for 30 min. The beads were pelleted by centrifugation for 2 min.in a Picofuge (Stratgene, Catalog #400550) at 2000×g and thesupernatants transferred to new 1.5 mL tubes. Twenty μg of anti-Flk-1antibody (Santa Cruz Biotechnology, Catalog #sc-504) was added to eachtube, and the tubes were incubated overnight (16-18 hr.) at 4 C on arotator to immunoprecipitate KDR. The next day 40 μl of proteinA-sepharose beads were added to the tubes that were then incubated 4 Cfor 1 hr. on a rotator. The beads in each tube were subsequently washedthree times by centrifuging for 2 min. in a Picofuge, discarding thesupernatant, and dispersing the beads in 1 mL freshly added TBST buffer(20 mM Tris base pH 7.5, 137 mM NaCl, and 0.1% Tween 20). Aftercentrifuging and removing the liquid from the last wash, 40 μl ofLaemmli SDS-PAGE sample buffer (Bio-Rad, Catalog #161-0737) was added toeach tube and the tubes were capped and boiled for 5 min. After cooling,the beads in each tube were pelleted by centrifuging and thesupernatants containing the immunoprecipitated KDR were transferred tonew tubes and used immediately or frozen and stored at −70 C for lateranalysis.

Detection of phosphorylated KDR as well as total KDR in theimmunoprecipitates was carried out by immunoblot analysis. Half (20 μL)of each immunoprecipitate was resolved on a 7.5% precast Ready Gel(Bio-Rad, Catalog #161-1154) by SDS-PAGE according to the method ofLaemmli (Nature, 227:680-685 (1970)).

Using a Bio-Rad mini-Protean 3 apparatus (Catalog #165-3302), theresolved proteins in each gel were electroblotted to a PVDF membrane(Bio-Rad, Cat. No. 162-0174) in a Bio-Rad mini Trans-Blot cell (Catalog#170-3930) in CAPS buffer (10 mM CAPS, Sigma Catalog #C-6070, 1% ACSgrade methanol, pH 11.0) for 2 hr. at 140 mA according to the method ofMatsudaira (J. Biol. Chem., 262:10035-10038 (1987)). Blots were blockedat room temperature in 5% Blotto-TBS (Pierce Catalog #37530) pre-warmedto 37° C. for 2 hr. The blots were first probed with ananti-phosphotyrosine antibody (Transduction Labs, Catalog #P11120),diluted 1:200 in 5% Blotto-TBS with 0.1% Tween 20 added for 2 hr. atroom temp. The unbound antibody was removed by washing the blots fourtimes with D-PBS containing 0.1% Tween 20 (D-PBST), 5 min. per wash.Subsequently, blots were probed with an HRP-conjugated sheep anti-mouseantibody (Amersham Biosciences Catalog #NA931) diluted 1:25,000 in 5%Blotto-TBS with 0.1% Tween 20 added for 1 hr. at room temp., and washedfour times with D-PBST. Finally, the blots were incubated with 2 mL of achemiluminescent substrate (ECL Plus, Amersham Catalog #RPN2132) spreadon top for 2 min., drip-drained well, placed in plastic sheet protector(C-Line Products, Catalog #62038), and exposed to X-ray film (KodakBioMax ML, Cat No. 1139435) for varying lengths of time to achieveoptimal contrast.

To confirm that similar amounts of KDR were compared in the assay, theblots were stripped by incubating for 30 min. at 37° C. in TBST with itspH adjusted to 2.4 with HCl, blocked for 1 hr. at room temp. with 5%Blotto-TBS with 0.1% Tween 20 (Blotto-TBST), and reprobed with ananti-Flk-1 polyclonal antibody (Catalog #sc-315 from Santa CruzBiotech), 1:200 in 5% Blotto-TBST with 1% normal goat serum (Life TechCatalog #16210064) for 2 hr. at room temp. The unbound antibody wasremoved by washing the blots four times with D-PBST, 5 min. per wash.Subsequently, the blots were probed with an HRP-conjugated donkeyanti-rabbit secondary antibody (Amersham Biosciences Catalog #NA934)diluted 1:10,000 in 5% Blotto-TBST for 1 hr. at room temp., and washedfour times with D-PBST. Finally, the blots were incubated with 2 mL ofchemiluminescent substrate and exposed to X-ray film as described above.

Results:

Immunoblots of KDR immunoprecipitates prepared from HUVECs with andwithout prior VEGF stimulation demonstrated that activated(phosphorylated) KDR could be detected when the HUVECs were stimulatedwith VEGF. An anti-phosphotyrosine antibody (PY-20) detected nophosphorylated proteins close to the migration position of KDR fromunstimulated HUVECs on the blots, but after five minutes of VEGFstimulation, an intense band was consistently observed at the expectedlocation (FIG. 11, upper panel). When the blots were stripped of boundantibodies by incubation in acidic solution then reprobed with ananti-KDR antibody (sc-315), the identity of the phosphorylated proteinband was confirmed to be KDR. Moreover, it was observed thatimmunoprecipitates from unstimulated HUVECs contained about as muchtotal KDR as immunoprecipitates from VEGF-stimulation HUVECs (FIG. 11,lower panel).

The foregoing results indicate that the phosphorylated KDR detected wasformed from pre-existing KDR through autophosphorylation of KDR dimersresulting from VEGF binding, as five minutes is not enough time tosynthesize and process a large glycosylated cell-surface receptor suchas KDR.

The ability of this assay to detect agents capable of blocking the VEGFactivation of KDR was assessed by adding a series of compounds to HUVECsin combination with VEGF and measuring KDR phosphorylation with theimmunoblot assay described above. As negative and positive controls,immunoprecipitates from unstimulated HUVECs and from HUVECs stimulatedwith VEGF in the absence of any test compounds were also tested in everyassay. When a neutralizing anti-KDR antibody (Catalog #AF-357 from R&DSystems) was combined with the VEGF, the extent of KDR phosphorylationwas greatly reduced (FIG. 12, upper panel), indicating that the antibodywas able to interfere with the ability of VEGF to bind to and activateKDR. This result was expected since the ability of the antibody to blockVEGF-induced DNA synthesis is part of the manufacturer's quality controltesting of the antibody. Re-probing the blot with an anti-KDR antibody(FIG. 12, lower panel) indicated that slightly less total KDR waspresent in the VEGF+antibody-treated lane (+V+α-KDR) relative to theVEGF-only-treated lane (+V), but the difference was not great enough toaccount for the much lower abundance of phosphorylated KDR in theantibody-treated lane.

To assess the potency of a linear KDR-binding peptide(AFPRFGGDDYWIQQYLRYTD, SEQ ID NO: 140) identified by phage display, theassay was repeated with a synthetic peptide containing the KDR-bindingsequence, Ac-AQAFPRFGGDDYWIQQYLRYTDGGK-NH₂ (SEQ ID NO: 306) in thepresence of VEGF. SEQ ID NO: 306 was able to inhibit the VEGF-inducedphosphorylation of KDR. Re-probing the blot for total KDR showed thatthere is even more total KDR in the VEGF+SEQ ID NO: 306-treated cells(+V+SEQ ID NO: 306) than in the VEGF only-treated cells (+V) (FIG. 13,lower panel). Thus, it is clear that the decreased phosphorylation ofKDR in the presence of SEQ ID NO: 306 is not due to differential sampleloading, but rather the ability of the polypeptide to inhibitVEGF-activation of KDR.

Repeating the foregoing assay, the following polypeptides demonstratedat least a 50% inhibition of VEGF-induced KDR phosphorylation at 10 μM:

(SEQ ID NO: 269) Ac-AGWIECYHPDGICYHFGTGGGK-NH₂ (SEQ ID NO: 267)Ac-AGWLECYAEFGHCYNFGTGGGK-NH₂ (SEQ ID NO: 294)Ac-GDSRVCWEDSWGGEVCFRYDPGGGK-NH₂ (SEQ ID NO: 366 having a blocked K)Ac-GDWWECK(ivDde)REEYRNTTWCAWADPGGGK-NH₂ (SEQ ID NO: 301)Ac-GDPDTCTMWGDSGRWYCFPADPGGGK-NH₂ (SEQ ID NO: 305)Ac-AQEPEGYAYWEVITLYHEEDGDGGK-NH₂ (SEQ ID NO: 306)Ac-AQAFPRFGGDDYWIQQYLRYTDGGK-NH₂ (SEQ ID NO: 307)Ac-AQGDYVYWEIIELTGATDHTPPGGK-NH₂.

SEQ ID NOS: 269 and 294 were the most potent compounds in the assay,producing at least a 50% inhibition of VEGF-induced KDR phosphorylationat 1 μM.

The following peptides were tested in the assay and did not producesignificant inhibition of KDR activation at 10 μM:

(SEQ ID NO: 264) Ac-AGPK(ivDde)WCEEDWYYCMITGTGGGK-NH₂ (SEQ ID NO: 314)Ac-GSDHHCYLHNGQWICYPFAPGGGK-NH₂ (SEQ ID NO: 293)Ac-GDYPWCHELSDSVTRFCVPWDPGGGK-NH₂ (SEQ ID NO: 295)Ac-GDDHMCRSPDYQDHVFCMYWDPGGGK-NH₂ (SEQ ID NO: 296)Ac-GDPPLCYFVGTQEWHHCNPFDPGGGK-NH₂ (SEQ ID NO: 299)Ac-GDGSWCEMRQDVGK(ivDde)WNCFSDDPGGGK-NH₂ (SEQ ID NO: 331)Ac-AQRGDYQEQYWHQQLVEQLK(ivDde)LLGGGK-NH₂ (SEQ ID NO: 303)Ac-GDNWECGWSNMFQK(ivDde)EFCARPDPGGGK-NH₂ (SEQ ID NO: 367)Ac-AGPGPCK(ivDde)GYMPHQCWYMGTGGGK-NH₂ (SEQ ID NO: 322)Ac-AGYGPCAEMSPWLCWYPGTGGGK-NH₂.

In addition, tetrameric complexes of biotinylated derivatives of SEQ IDNOS: 294 and 277 (prepared as described above) produced at least a 50%inhibition of VEGF-induced KDR phosphorylation at 10 nM.

Example 10 Binding of Tc-Labeled SEQ ID NO: 339 to KDR-Transfected 293HCells

The ability of Tc-labeled SEQ ID NO: 339 to bind KDR was assessed usingKDR-transfected 293H cells. Tc-labeled SEQ ID NO: 277 (i.e.,Ac-AGPTWCEDDWYYCWLFGTGGGK(N,N-dimethyl-Gly-Ser-Cys-Gly-di(aminodioxaocta-))-NH₂)bound significantly better to KDR transfected 293H cells than to mocktransfected 293H cells and binding increased with concentration ofTc-labeled SEQ ID NO: 339 in a linear manner.

Preparation of Peptidic Chelate for Binding to Tc by SPPS (FIG. 35)

To a 250 mL of SPPS reaction vessel was added 6.64 mmol ofH-Gly-2-Cl-trityl resin (0.84 mmol/g, Novabiochem). It was swelled in 80mL of DMF for 1 h. For each coupling cycle the resin was added 26.6 mmolof DIEA, 26.6 mmol of a Fmoc-amino acid in DMF (EM Science), 26.6 mmolof HOBT (Novabiochem) in DMF, and 26.6 mmol of DIC. The total volume ofDMF was 80 mL. The reaction mixture was shaken for 4 h. The resin thenwas filtered and washed with DMF (3×80 mL). A solution of 20% piperidinein DMF (80 mL) was added to the resin and it was shaken for 10 min. Theresin was filtered and this piperidine treatment was repeated. The resinfinally was washed with DMF (3×80 mL) and ready for next coupling cycle.At the last coupling cycle, N,N-dimethyl glycine (Aldrich) was coupledusing HATU/DIEA activation. Thus, to a suspension of N,N-dimethylglycine (26.6 mmol) in DMF was added a solution of 26.6 mmol of HATU(Perseptive Biosystems) in DMF and 53.1 mmol of DIEA. The clear solutionwas added to the resin and shaken for 16 h. Following the synthesis, theresin was filtered and washed with DMF (3×80 mL), CH₂Cl₂ (3×80 mL) anddried. The resin was mixed with 80 mL of AcOH/CF₃CH₂OH/DCM (1/1/8,v/v/v) and shaken for 45 min. The resin was filtered and the filtratewas evaporated to a paste. Purification of the crude material by silicagel chromatography using 25% MeOH/DCM afforded 2.0 g of the finalproduct.

Coupling of the Peptidic Chelate to the Peptide (Fragment Coupling)

Diisopropylcarbodiimide (0.0055 mmol) was added to a mixture of purifiedMe₂N-Gly-Cys-(Trt)-Ser(tBu)-Gly-OH and hydroxybenzotriazole (0.0055mmol) in DMF (0.25 mL), and the mixture was stirred at RT for 6 h. Thepeptide (0.005 mmol) in DMF (0.25 mL) was then added to the reactionmixture, and stirring was continued for an additional 6 h. DMF wasremoved under vacuum and the residue was treated with reagent B andstirred for 3 h. TFA was removed under reduced pressure and the residuewas purified by preparative HPLC using acetonitrile-water containing0.1% TFA. Fractions containing the pure product were collected andfreeze dried to yield the peptide. The peptide was characterized byES-MS and the purity was determined by RP-HPLC (acetonitrile-water/0.1%TFA) gradient.

Synthesis of ^(99m)Tc-Labeled SEQ ID NO: 339

A stannous gluconate solution was prepared by adding 2 mL of a 20 μg/mLSnCl₂.2H₂O solution in nitrogen-purged 1N HCl to 1.0 mL ofnitrogen-purged water containing 13 mg of sodium glucoheptonate. To a 4mL autosampler vial was added 20-40 μl (20-40 μg) of SEQ ID NO: 339ligand dissolved in 50/50 ethanol/H₂O, 6-12 mCi of ^(99m)TcO₄ ⁻ insaline and 100 μl of stannous glucoheptonate solution. The mixture washeated at 100° C. for 22 min. The resulting radiochemical purity (RCP)was 10-47% when analyzed using a Vydac C18 Peptide and Protein columnthat was eluted at a flow rate of 1 mL/min. with 66% H₂O (0.1% TFA)/34%ACN (0.085% TFA). The reaction mixture was purified by HPLC on a VydacC18 column (4.6 mm×250 mm) at a flow rate of 1 mL/min., using 0.1% TFAin water as aqueous phase and 0.085% TFA in acetonitrile as the organicphase. The following gradient was used: 29.5% org. for 35 min., ramp to85% over 5 min., hold for 10 min. The fraction containing ^(99m)Tc SEQID NO: 339 was collected into 500 μl of a stabilizing buffer containing5 mg/mL ascorbic acid and 16 mg/mL hydroxypropyl-α-cyclodextrin in 50 mMphosphate buffer. The mixture was concentrated using a speed vacuumapparatus to remove acetonitrile, and 200 μl of 0.1% HSA in 50 mM pH 5citrate buffer was added. The resulting product had an RCP of 100%.Prior to injection into animals, the compound was diluted to the desiredradioconcentration with normal saline.

Transfection of 293H Cells

293H cells were transfected using the protocol described above.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. #354640). The left half of the plates (48 wells) weremock-transfected (with no DNA) and the right half of the plate wastransfected with KDR cDNA. The cells were 80-90% confluent at the timeof transfection and completely confluent the next day, at the time ofthe assay; otherwise the assay was aborted.

Preparation of Opti-MEMI Media with 0.1% HSA

Opti-MEMI was obtained from Invitrogen (cat. #11058-021) and human serumalbumin (HSA) was obtained from Sigma (cat. # A-3782). To prepareopti-MEMI media with 0.1% HSA, 0.1% w/v HSA was added to opti-MEMI,stirred at room temperature for 20 min. and then filter sterilized using0.2 μm filter.

Preparation of Tc-Labeled SEQ ID NO: 339 Dilutions for the Assay

Stock solution of Tc-labeled SEQ ID NO: 339 (117 μCi/mL) was diluted1:100, 1:50, 1:25 and 1:10 in opti-MEMI with 0.1% HSA to providesolutions with final concentration of 1.17, 2.34, 4.68 and 11.7 μCi/mLof Tc-labeled SEQ ID NO: 339.

Assay to Detect the Binding of Tc-Labeled SEQ ID NO: 339

Cells were used 24 hours after transfection, and to prepare the cellsfor the assay, they were washed once with 100 μl of room temperatureopti-MEMI with 0.1% HSA. After washing, the opti-MEMI with 0.1% HSA wasremoved from the plate and replaced with 70 μl of 1.17, 2.34, 4.68 and11.7 μCi/mL of Tc-labeled SEQ ID NO: 339 (prepared as above). Eachdilution was added to three separate wells of mock- and KDR-transfectedcells. After incubating at room temperature for 1 hour, the plates weretransferred to 4° C. for 15 minutes and washed 5 times with 100 μl ofcold binding buffer (opti-MEMI with 0.1% HSA), gently blotted dry andchecked under a microscope for cell loss. 100 μl of solubilizingsolution (2% Triton X-100, 10% Glycerol, 0.1% BSA) was added to eachwell and the plates were incubated at 37° C. for 10 minutes. Thesolubilizing solution in each well was mixed by pipeting up and down,and transferred to 1.2 mL tubes. Each well was washed once with 100 μlof solubilizing solution and the washes were added to the corresponding1.2 mL tube. Each 1.2 mL tube was then transferred to a 15.7×100 cm tubeto be counted in an LKB Gamma Counter using program 12 (Tc-window for 20sec).

Binding of Tc-Labeled SEQ ID NO: 339 to KDR Transfected Cells

The ability of Tc-labeled SEQ ID NO: 339 to specifically bind to KDR wasassessed using transiently transfected 293H cells.

As shown in FIG. 14, Tc-labeled SEQ ID NO: 339 bound significantlybetter to KDR transfected 293H cells as compared to mock transfected293H cells. To calculate specific binding to KDR, the binding ofTc-labeled SEQ ID NO: 339 polypeptide to mock-transfected cells wassubtracted from the binding to KDR-transfected cells. A linear increasein the specific binding of Tc-labeled SEQ ID NO: 339 to KDR was observedwith increasing concentration of Tc-labeled SEQ ID NO: 339 (FIG. 96).Linear binding was not surprising because concentration of Tc-labeledSEQ ID NO: 339 was only ˜100 pM (even at the highest concentration, 11.7μCi/mL, tested in the assay), which is far below the K_(D) value of ˜3-4nM of SEQ ID NO: 277 (as calculated using avidin HRP assay), so nosaturation of binding would be expected.

Example 11 Preparation of Peptides and Dimeric Peptide Construction

The following methods were used for the preparation of individualpeptides and dimeric peptide constructs described in the followingExamples (11-15).

Automated Peptide Synthesis

Peptide synthesis was carried out on a ABI-433A Synthesizer (AppliedBiosystems Inc., Foster City, Calif.) on a 0.25 mmol scale using theFastMoc protocol. In each cycle of this protocol preactivation wasaccomplished by dissolution of 1.0 mmol of the requisite dry N^(α)-Fmocside-chain protected amino acid in a cartridge with a solution of 0.9mmol of HBTU, 2 mmol of DIEA, and 0.9 mmol of HOBt in a DMF-NMP mixture.The peptides were assembled on NovaSyn TGR (Rink amide) resin(substitution level 0.2 mmol/g). Coupling was conducted for 21 min. Fmocdeprotection was carried out with 20% piperidine in NMP. At the end ofthe last cycle, the N-terminal Fmoc group was removed and the fullyprotected resin-bound peptide was acetylated using aceticanhydride/DIEA/HOBt/NMP.

Cleavage, Side-chain Deprotection and Isolation of Crude Peptides

Cleavage of the peptides from the resin and side-chain deprotection wasaccomplished using Reagent B for 4.5 h at ambient temperature. Thecleavage solutions were collected and the resins were washed with anadditional aliquot of Reagant B. The combined solutions wereconcentrated to dryness. Diethyl ether was added to the residue withswirling or stirring to precipitate the peptides. The liquid phase wasdecanted, and solid was collected. This procedure was repeated 2-3 timesto remove impurities and residual cleavage cocktail components.

Cyclization of Di-Cysteine Peptides

The crude ether-precipitated linear di-cysteine containing peptides werecyclized by dissolution in water, mixtures of aqueous acetonitrile (0.1%TFA), aqueous DMSO or 100% DMSO and adjustment of the pH of the solutionto 7.5-8.5 by addition of aqueous ammonia, aqueous ammonium carbonate,aqueous ammonium bicarbonate solution or DIEA. The mixture was stirredin air for 16-48 h, acidified to pH 2 with aqueous trifluoroacetic acidand then purified by preparative reverse phase HPLC employing a gradientof acetonitrile into water. Fractions containing the desired materialwere pooled and the purified peptides were isolated by lyophilization.

Preparation of Peptides Containing Linkers

In a typical experiment, 400 mg of the resin-bound peptide bearing anivDde-protected lysine) was treated with 10% hydrazine in DMF (2×20 mL).The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). The resin wasresuspended in DMF (10 mL) and treated withFmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC(0.4 mmol) and DIEA (0.8 mmol) with mixing for 4 h. After the reaction,the resin was washed with DMF (2×10 mL) and with DCM (1×10 mL). Theresin was then treated with 20% piperidine in DMF (2×15 mL) for 10 mineach time. The resin was washed and the coupling withFmoc-8-amino-3,6-dioxaoctanoic acid and Fmoc protecting group removalwere repeated once more.

The resulting resin-bound peptide with a free amino group was washed anddried and then treated with reagent B (20 mL) for 4 h. The mixture wasfiltered and the filtrate concentrated to dryness. The residue wasstirred with ether to produce a solid, which was washed with ether anddried. The solid was dissolved in anhydrous DMSO and the pH adjusted to7.5 with DIEA. The mixture was stirred for 16 h to effect the disulfidecyclization and the reaction was monitored by analytical HPLC. Aftercompletion of the cyclization, the reaction mixture was diluted with 25%acetonitrile in water and applied directly to a reverse phase C-18column. Purification was effected using a gradient of acetonitrile intowater (both containing 0.1% TFA). Fractions were analyzed by HPLC andthose containing the pure product were combined and lyophilized toprovide the required peptide.

Preparation of Biotinylated Peptides Containing Linkers

In a typical experiment, 400 mg of the resin-bound peptide bearing anivDde-protected lysine, was treated with 10% hydrazine in DMF (2×20 mL).The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). The resin wasresuspended in DMF (10 mL) and treated withFmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC(0.4 mmol) and DIEA (0.8 mmol) with mixing for 4 h. After the reaction,the resin was washed with DMF (2×10 mL) and with DCM (1×10 mL). Theresin was then treated with 20% piperidine in DMF (2×15 mL) for 10 mineach time. The resin was washed and the coupling withFmoc-8-amino-3,6-dioxaoctanoic acid and removal of the Fmoc protectinggroup were repeated once more.

The resulting resin-bound peptide with a free amino group was treatedwith a solution of Biotin-NHS ester (0.4 mmol, 5 equiv.) and DIEA (0.4mmol, 5 equiv.) in DMF for 2 h. The resin was washed and dried asdescribed previously and then treated with Reagent B (20 mL) for 4 h.The mixture was filtered and the filtrate concentrated to dryness. Theresidue was stirred with ether to produce a solid that was collected,washed with ether, and dried. The solid was dissolved in anhydrous DMSOand the pH adjusted to 7.5 with DIEA. The mixture was stirred for 4-6 hto effect the disulfide cyclization, which was monitored by HPLC. Uponcompletion of the cyclization, the reaction mixture was diluted with 25%acetonitrile in water and applied directly to a reverse phase C-18column. Purification was effected using a gradient of acetonitrile intowater (both containing 0.1% TFA). Fractions were analyzed by HPLC andthose containing the pure product were collected and lyophilized toprovide the required biotinylated peptide.

Preparation of Dota-Conjugated Peptides for Labeling with SelectedGadolinium or Indium Isotopes

In a typical experiment, 400 mg of the resin-bound peptide bearing anN^(ε)-ivDde-protected lysine moiety was treated with 10% hydrazine inDMF (2×20 mL). The resin was washed with DMF (2×20 mL) and DCM (1×20mL). The resin was resuspended in DMF (10 mL) and treated withFmoc-8-amino-3,6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC(0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. After the reaction, theresin was washed with DMF (2×10 mL) and with DCM (1×10 mL). The resinwas then treated with 20% piperidine in DMF (2×15 mL) for 10 min eachtime. The resin was washed and the coupling withFmoc-8-amino-3,6-dioxaoctanoic acid and removal of the Fmoc protectinggroup were repeated once. The resulting resin-bound peptide with a freeamino group was resuspended in DMF (10 mL) and treated with a solutionof 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid,-1,4,7-tris-t-butyl ester (DOTA-tris-t-butyl ester, 0.4 mmol, 5 equiv.),HOBt (0.4 mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) in DMF (10 mL) withmixing for 4 h. Upon completion of the reaction, the resin was washedwith DMF (2×10 mL) and with DCM (1×10 mL) and treated with Reagent B (20mL) for 4 h. The mixture was filtered and the filtrate concentrated todryness. The residue was stirred in ether to produce a solid that wascollected, washed with ether, and dried. The solid was dissolved inanhydrous DMSO and the pH adjusted to 7.5 with DIEA. The mixture wasstirred for 16 h to effect the disulfide cyclization, which wasmonitored by HPLC. Upon completion of the cyclization, the mixture wasdiluted with 25% acetonitrile in water and applied directly to a reversephase C-18 HPLC column. Purification was effected using a gradient ofacetonitrile into water (both containing 0.1% TFA). Fractions wereanalyzed by HPLC and those containing the pure product were combined andlyophilized to provide the required biotinylated peptide.

The following monomeric peptides of Table 11 were prepared by the abovemethods, “PnAO6”, as used herein, refers to3-(2-amino-3-(2-hydroxyimino-1,1-dimethyl-propylamino)-propylamino)-3-methyl-butan-2-oneoxime.

TABLE 11 Sequence or Structure of MonomericPeptides and Peptide Derivatives SEQ. ID NO: Structure or Sequenceor dimer Ac-AGPTWCEDDWYYCWLFGTGGGK(BiotinJJ-K)-NH₂ 277(Ac-AGPTWCEDDWYYCWLFGTGGGKK(BiotinJJ-)-NH₂₎ 373Ac-AGPTWCEDDWYYCWLFGTJK(DOTAJJ-K)-NH₂ 493Ac-AGPTWCEDDWYYCWLFGTJK(JJ)-NH₂ 493Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ 373Ac-VCWEDSWGGEVCFRYDPGGGK(Biotin-JJK)-NH₂ 337(Ac-VCWEDSWGGEVCFRYDPGGGKK(Biotin-JJ)-NH₂) 494Ac-VCWEDSWGGEVCFRYDPGGGK(JJ)-NH₂ 337Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(J)-NH₂ 356 Seq 12 derivativeAc-AQDWYYDEILSMADQLRHAFLSGGGGGKK(ivDde) 495Application seq 12 derivative Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH₂ 294Seq 5 derivative Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH₂ Seq 5 deriv294/D10 Ac-AGPTWCEDDWYYCWLFGTGGGK[(PnAO6- 277/D10C(═O)(CH₂)₃C(═O)—K]—NH₂ A Seq 11 derivativeAc-AGPTWCEDDWYYCWLFGTGGGK[(DOTA-JJK(iV-Dde)]- 277/D11NH₂ A Seq 11 derivative Ac-AGPTWCEDDWYYCWLFGTGGGK[(PnAO6- 476/D12C(═O)(CH₂)₃C(═O))K]—NH₂ A Seq 11 derivativeAc-VCWEDSWGGEVCFRYDPGGGK-NH₂ A Seq 5 derivative 337/D12specifically: Seq 5 residues 5-25Ac-AGPTWCEDDWYYCWLFGTGGGK[K(BOA)]-NH₂ Seq 11 277/D13 derivativeAc-AQDWYYDEILSMADQLRHAFLSGGGGGK[PnAO6- 356/D14C(═O)(CH₂)₃C(═O)—K(iV-Dde)]-NH₂ Application seq 12 derivativeAc-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH₂ Seq 5 deriv 294/D15 linker = GlutAc-AGPTWCEDDWYYCWLFGTGGGK-[PnAO6- 277/D16C(═O)(CH₂)₃C(═O)—K]—NH₂ A Seq 11 derivative, new sequenceAc-AQDWYYEILJGRGGRGGRGGK[K(ivDde)]-NH₂ 496/D17 A Seq 12 (1-9) derivativeAc-APGTWCDYDWEYCWLGTFGGGK[(6PnAO- 497/D18C(═O)(CH₂)₃C(═O)—K]—NH₂ A scrambled Seq 11 derivative used as a control.Ac-GVDFRCEWSDWGEVGCRSPDYGGGK(JJ)-NH₂ A 489/D18scrambled Seq 5 derivative.Ac-AGPTWCEDDWYYCWLFGTGGGK(Biotin-K)—NH₂, A Seq 294/D19 11 derivativeJJAGPTWCEDDWYYCWLFGTGGGK(iV-Dde)-NH₂ (SEQ ID 277/D20 NO: 277)JJVCWEDSWGGEVCFRYDPGGG-NH₂ 370/D20 JJAGPTWCEDDWYYCWLFGTGGGK(iV-Dde)-NH₂277/D21 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(SATA)]-NH₂ 373/D22Ac-AGPTWCEDDWYYCWLFGTGGGK[SATA-JJ-K]-NH₂ 339/D23Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH₂ 294/D24H₂N-AGPTWCEDDWYYCWLFGTGGGK[K(iV-Dde)]-NH₂ 373/D25Ac-AGPTWCEDDWYYCWLFGTGGGK{Biotin-JJK[NH₂- 339/D26Ser(GalNAc(Ac)₃-alpha-D)-Gly-Ser(GalNAc(Ac)₃-alpha-D]}-NH₂Ac-VCWEDSWGGEVCFRYDPGGGK(NH₂-Ser(GalNAc(Ac)₃- 337/D26alpha-D)-Gly-Ser(GalNAc(Ac)₃-alpha-D)-NH₂Ac-GSPEMCMMFPFLYPCNHHAPGGGK[(PnAO6)- 482/D27C(═O)(CH₂)₃C(═O)—K]}-NH₂ A modified cMet Binding Sequence

Example 12 Preparation of Homodimeric and Heterodimeric Constructs

The purified peptide monomers mentioned above in Example 8 were used inthe preparation of various homodimeric and heterodimeric constructs.

Preparation of Homodimer-Containing Constructs

To prepare homodimeric compounds, half of the peptide needed to preparethe dimer was dissolved in DMF and treated with 10 equivalents ofglutaric acid bis-N-hydroxysuccinimidyl ester. The progress of thereaction was monitored by HPLC analysis and mass spectroscopy. Atcompletion of the reaction, the volatiles were removed in vacuo and theresidue was washed with ethyl acetate to remove the unreacted bis-NHSester. The residue was dried, re-dissolved in anhydrous DMF and treatedwith another half portion of the peptide in the presence of 2equivalents of DIEA. The reaction was allowed to proceed for 24 h. Thismixture was applied directly to a YMC reverse phase HPLC column andpurified by elution with a linear gradient of acetonitrile into water(both containing 0.1% TFA).

Preparation of Heterodimer-Containing Constructs

In the case of heterodimers, one of the monomers (“A”) was reacted withthe bis-NHS ester of glutaric acid and after washing off the excess ofbis-NHS ester (as described for the homodimeric compounds), the secondmonomer (“B”) was added in the presence of DIEA. After the reaction themixture was purified by preparative HPLC. Typically, to a solution ofglutaric acid bis N-hydroxysuccinimidyl ester (0.02 mmol, 10equivalents) in DMF (0.3 mL) was added a solution of peptide “A” andDIEA (2 equiv) in DMF (0.5 mL) and the mixture was stirred for 2 h. Theprogress of the reaction was monitored by HPLC analysis and massspectroscopy. At completion of the reaction, the volatiles were removedin vacuo and the residue was washed with ethyl acetate (3×1.0 mL) toremove the unreacted bis-NHS ester. The residue was dried, re-dissolvedin anhydrous DMF (0.5 mL) and treated with a solution of peptide “B” andDIEA (2 equiv) in DMF (0.5 mL) for 24 h. The mixture was diluted withwater (1:1, v/v) and applied directly to a YMC C-18 reverse phase HPLCcolumn and purified by elution with a linear gradient of acetonitrileinto water (both containing 0.1% TFA). Fractions were analyzed byanalytical HPLC and those containing the pure product were combined andlyophilized to obtain the required dimer. The dimers depicted in FIGS.36-63 were prepared by this method (structure, name, compound referencenumber as described in the “Brief Description of the Drawings”).

For the preparation of the dimer D5, after the coupling reaction of theindividual peptides, 50 μL of hydrazine was added to the reactionmixture (to expose the lysine N^(E)-amino group) and the solution wasstirred for 2 min. The reaction mixture was diluted with water (1.0 mL)and the pH was adjusted to 2 with TFA. This was then purified by themethod described above.

The HPLC analysis data and mass spectral data for the dimeric peptidesare given in Table 12 below.

TABLE 12 Analytical Data for Homodimeric and Heterodimeric PeptideConstructs HPLC Analysis System Retention Time (System) Mass Spectraldata (API-ES, - ion) D1 8.98 min. (A) 1987.7 (M − 3H)/3, 1490.6 (M −4H)/4, 1192.3 (M − 5H)/5 D2 16.17 min (B) 2035.3 (M − 3H)/3, 1526.1 (M −4H)/4, 1220.7 (M − 5H)/5 D3 8.74 min (C) 1933.6 (M − 3H)/3, 1449.9 (M −4H)/4, 1159.4 (M − 5H)/5 D4 10.96 min (D) 2032.8 (M − 3H)/3 D5 6.57 min(E) 1816.2 (M − 3H)/3, 1361.8 (M − 4H)/4, 1089.4 (M − 5H)/5, 907.7 (M −6H)/6 D6 D7 D8 4.96 min; (F) 2379.3 [M − 3H]/3 D9 5.49 min; (G) 2146.4[M − 3H]/3 D10 5.44 min; (H) 2082.7 [M − 3H]/3, 1561.7 [M − 4H]/4,1249.1 [M − 5H]/5, 1040.7 [M − 6H]/6 D11 7.23 min; (E) 2041.8 [M −3H]/3, 1531.1 [M − 4H]/4, 1224.6 [M − 5H]/5 D12 5.84 min; (H) 1877.1 [M− 3H]/3, 1407.6 [M − 4H]/4, 1125.9 [M − 5H]/5, 938.1 [M − 6H]/6. D135.367 min; (E) 1965.3 [M − 3H]/3, 1473.8 [M − 4H]/4, 1178.8 [M − 5H]/5,982.2 [M − 6H]/6 D14 4.78 min; (I) 2275.0 [M − 3H]/3, 1362.8 [M − 5H]/5D15 5.41 min; (H) 1561.3 [M − 4H]/4, 1249.1 [M − 5H]/5, 1040.8 [M −6H]/6, 891.8 [M − 7H]/7. D16 5.44 min; (J) 2150.8 [M − 3H]/3, 1613.1 [M− 4H]/4, 1289.9 [M − 5H]/5, 1074.8 [M − 6H]/6, 920.9 [M − 7H]/7. D174.78 min; (K) 1789.4 [M − 3H]/3, 1347.7 [M − 4H]/4. D18 4.74 min; (L)2083.1 [M − 3H]/3, 1562.7 [M − 4H]/4, 1249.5 [M − 5H]/5. D19 7.13 min;(O) 1891.9 [M − 3H]/3, 1418.4 [M − 4H]/4, 1134.8 [M − 5H]/5, 945.5 [M −6H]/6. D20 9.7 min; (P) 2700.4 [M − 2H]/2, 1799.3[M − 3H]/3 D21 6.1 min;(P) 2891.3 [M − 2H]/2, 1927.2[M − 3H]/3, 1445.1 [M − 4H]/4, 1155.8 [M −5H]/5. D22 6.23 min; (Q) 1994.4 [M − 3H]/3, 1495.7 [M − 4H]/4, 1196.3 [M− 5H]/5 D23 7.58 min; (J) 1854.4 [M − 3H]/3, 1390.8 [M − 4H]/4, 1112.7[M − 5H]/5, 927 [M − 6H]/6 D24 8.913 min; (R) 1952.1 [M − 3H]/3, 1463.4[M − 4H]/4, 1171.1 [M − 5H]/5, 975.3 [M − 6H]/6 D25 5.95 min; (E) 1954.9[M − 3H]/3, 1466.1 [M − 4H]/4, 1172.4 [M − 5H]/5, 976.8 [M − 6H]/6. D266.957 min; (S) 1759.1 [M − 3H]/3, 1319.6 [M − 4H]/4, 1055.1 [M − 5H]/5D27 5.5 min; (M) 2317.6 [M − 3H]/3, 1737.2[M − 4H]/4, 1389.3[M − 5H]/5,1157.7 [M − 6H]/6 D30 4.29 min (T) [M + H]: 5782.3, [M + 4H]/4: 1146.6,[M + 5H]/5: 1157.4, [M + 6H]/6: 964.7 D31 6.6 min (U) [M − 3H]/3:2045.3. Monomer 6.0 min (U) [M − 2H]/2: 1307.4 Compound 2 Monomer 5.3min (U) [M − 2H]/2: 1307.4 Compound 4 (SEQ ID NO: 374-related sequence)

TABLE 13 Dimer sequences and linkers Dimer # Sequence D1Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277)[(Biotin-JJK- (FIG. 36)(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337))-NH)CONH₂ ]—NH₂) D2Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277) [(Biotin-JJK- (FIG. 37)(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-AGPTWCEDDWYYCWLFGTJK(SEQ ID NO: 493))-NH)CONH₂ ]—NH₂ D3Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)[(Biotin-JJK- (FIG. 38)(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337))-NH)CONH₂]—NH₂ D4Ac-AGPTWCEDDWYYCWLFGTJK(SEQ ID NO: 338)[DOTA-JJK- (FIG. 39)(O═)C(CH₂)₃C(═O)-JJ-NH(CH₂)₄—(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337))-NH)CONH₂]—NH₂ D5Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337) (JJ-C(═O)(CH₂)₃C(═O)—K—(FIG. 40) NH(CH₂)₄—(S)—CH((Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277))-NH)CONH₂)—NH₂ D6 GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 294)- (FIG. 63)AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO: 277) (see FIG. 63 for linkage) D7GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 294)- (FIG. 64)AGPKWCEEDWYYCMITGTGGGK (SEQ ID NO: 264) (see FIG. 64 for linkage) D8Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK{Ac- (FIG. 41)AQDWYYDEILSMADQLRHAFLSGGGGGK(J-Glut-)-NH₂}K(Biotin-JJ)-NH₂ D9Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK{[Ac- (FIG. 42)GDSRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut-)]-NH₂}K—NH₂ D10Ac-AGPTWCEDDWYYCWLFGTGGGK{[Ac- (FIG. 43)GDSRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut-NH(CH₂)₄—(S)—CH(PnAO6-Glut-NH)(C═O-)]-NH₂}—NH₂ D11 Ac-AGPTWCEDDWYYCWLFGTGGGK{[Ac- (FIG. 44)VCWEDSWEDSWGGEVCFRYDPGGGK[JJ-Glut-NH(CH₂)₄—(S)—CH(DOTA-JJ-NH—)(C═O)—]—NH₂}—NH₂D12 Ac-AGPTWCEDDYCWLFGTGGGK{[PnAO6-Glut-K(Ac- (FIG. 45)VCWEDSWGGEVCFRYDPGGGK(-C(═O)CH₂(OCH₂CH₂)₂OCH₂C(═O)—)—NH₂]}—NH₂ D13Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[JJ- (FIG. 46)Glut-K(BOA)]-NH₂}—NH₂: Dimer 13 (D13) D14Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK{PnAO6-Glut-K[Ac- (FIG. 47)GSDRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH₂]}—NH₂ D15Ac-AGPTWCEDDWYYCWLFGTGGGK{[[Ac- (FIG. 48)GDSRVCWEDSWGGEVCFRYDPGGGKJJ-Glut]-NH₂]—K(PnAO6-Glut)}—NH₂ D16Ac-AGPTWCEDDWYYCWLFGTGGGGK{PnAO6-Glut-K[Ac- (FIG. 49)GDSRVCWEDSWGGEVCFRYDPGGGK[-C(═O)CH₂O(CH₂CH₂O)₂CH₂C(═O)NH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NHC(═O)CH₂O(CH₂CH₂O)₂CH₂C(═O)—]—NH₂]}—NH₂D17 Ac-AQDWYYDEILJGRGGRGGRGGK{K[Ac- (FIG. 50)VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH₂]}—NH₂ D18Ac-AGPTWCDYDWEYCWLGTFGGGK{PnAO6-Glut-K[Ac- (FIG. 51)GVDFRCEWSDWGEVGCRSPDYGGGK(JJ-Glut)-NH₂]}—NH₂ D19Ac-AGPTWCEDDWYYCWLFGTGGGK{Biotin-K[Ac- (FIG. 52)VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH₂]}—NH₂ D20(-JJAGPTWCEDDWYYCWLFGTGGGGK-NH₂)-Glut- (FIG. 53)VCWEDSWGGEVCFRYDPGGG-NH₂ D21[-JJAGPTWCEDDWYYCWLFGTGGGGK(PnAO6-Glut)-NH₂]-Glut- (FIG. 54)VCWEDSWGGEVCFRYDPGGG-NH₂ D22 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK{JJ-Glut-JJ-(FIG. 55) AGPTWCEDDWYYCWLFTGGGK-NH₂}—NH₂ D23Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[JJ- (FIG. 56)Glut-K(SATA)]-NH₂}—NH₂ D24 Ac-AGPTWCEDDWYYCWLFGTGGGK{SATA-JJK[Ac-(FIG. 57) VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH₂]}—NH₂ D25Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac- (FIG. 58)GDSRVCWEDSWGGEVCFRYDPGGGK[JJ-Glut-NH(CH₂)4-(S)—CH(NH₂)C(═O)—]—NH₂}—NH₂D26 AGPTWCEDDWYYCWLFGTGGGGK{(-Glut-JJ-VCWEDSWGGEVCFRYDPGGG- (FIG. 59)NH₂)—K}—NH₂ D27 Ac-AGPTWCEDDWYYCWLFGTGGGGK{Ac- (FIG. 60)VCWEDSWGGEVCFRYDPGGGK[S(GalNAc(Ac)₃-alpha-D)-G-S(GalNAc(Ac)₃-alpha-D)-Glut-S(GalNAc(Ac)₃-alpha-D)-G-S(GalNAc(Ac)₃-alpha-D)-NH(CH₂)₄—(S)—CH(Biotin-JJNH-)C(═O)—]—NH₂}—NH₂ D28 AQEPEGYAYWEVITLYHEEDGDGGK (SEQ ID NO: 305)-(FIG. 61)AQAFPRFGGDDYWIQQYLRYTDGGK (SEQ ID NO: 306) (see FIG. 61 for linkage) D29AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO: 277)- (FIG. 62)VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 337) (see FIG. 62 for linkage) D30Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 337){[PnAO₆-Glut-K(-Glut-JJ- (FIG.NH(CH₂)₄—(S)—CH(Ac-AQDWYYDEILJGRGGRGGRGG(SEQ ID NO: 478)- 87C)NH)C(═O)NH₂]—NH2}—NH₂(see FIG. 87C) D31Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277)[Ac- (FIG.VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)[SGS-Glut-SGS—(S)—NH(CH₂)₄—CH(Biotin-88D) JJ-NH)—C(═O)]—NH₂]—NH₂(see FIG. 88D)HPLC Analysis Systems

System A: Column: YMC C-4 (4.6×250 mm); Eluents: A: Water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 25% B, linear gradient25-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System B: Column: YMC C-4 (4.6×250 mm); Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 25% B, linear gradient25-60% B in 20 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System C: Column: YMC C-4 (4.6×250 mm); Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 30% B, linear gradient30-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System D: Column: YMC C-4 (4.6×250 mm); Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient20-60% B in 10 min; flow rate: 2.0 mL/min; Detection: UV @ 220 nm.

System E: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, lineargradient 10-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System F: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: Acetonitrile (0.1% TFA); Elution: Initial condition, 30% B,Linear Gradient 30-70% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV@ 220 nm.

System G: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 30% B, lineargradient 30-75% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System H: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, lineargradient 20-52% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System I: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, lineargradient 10-65% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System J: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, lineargradient 20-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System K: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 5% B, lineargradient 5-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System L: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 5% B, lineargradient 5-65% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System M: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 15% B, lineargradient 15-50% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System N: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, lineargradient 20-80% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System O: Column: YMC-C18, 4.6×250 mm; Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 30% B, linear gradient30-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System P: Column: YMC-C18, 4.6×250 mm; Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient20-80% B in 20 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System Q: Column: YMC-C18, 4.6×250 mm; Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient20-60% B in 6 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System R: Column: YMC-C18, 4.6×250 mm; Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 25% B, linear gradient25-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.

System S: Column: YMC-C18, 4.6×100 mm; Eluents: A: water (0.1% TFA), B:ACN (0.1% TFA); Elution: initial condition, 10% B, linear gradient10-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm.

System T: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 5% B, lineargradient 5-65% B in 8 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

System U: Column: Waters XTerra, 4.6×50 mm; Eluents: A: water (0.1%TFA), B: ACN (0.1% TFA); Elution: initial condition, 15% B, lineargradient 15-50% B in 8 min; flow rate: 3.0 mL/min; detection: UV @ 220nm.

Example 13 Competition with ¹²⁵I-VEGF for binding to KDR on HUVECs andKDR-Transfected Cells

The following experiment assessed the ability of KDR-binding peptides tocompete with ¹²⁵I-labeled VEGF for binding to KDR expressed bytransfected 293H cells.

Protocol

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques. The cells were incubated with ¹²⁵I-VEGF in thepresence or absence of competing compounds (at 10 μM, 0.3 μM, and 0.03μM). After washing the cells, the bound radioactivity was quantitated ona gamma counter. The percentage inhibition of VEGF binding wascalculated using the formula [(Y1−Y2)×100/Y1], where Y1 is specificbinding to KDR-transfected 293H cells in the absence peptides, and Y2 isspecific binding to KDR-transfected 293H cells in the presence ofpeptide competitors. Specific binding to KDR-transfected 293H cells wascalculated by subtracting the binding to mock-transfected 293H cellsfrom the binding to KDR-transfected 293H cells.

Results

As shown in FIG. 15, all of the KDR-binding peptides assayed were ableto compete with ¹²⁵I-VEGF for binding to KDR-transfected cells. Theheterodimer (D1) was clearly the most effective at competing with¹²⁵I-VEGF, even over the two homodimers (D2 and D3), confirming thesuperior binding of D1.

Example 14 Receptor Activation Assay

The ability of KDR-binding peptides to inhibit VEGF induced activation(phosphorylation) of KDR was assessed using the following assay.

Protocol

Dishes of nearly confluent HUVECs were placed in basal medium lackingserum or growth factors overnight. The dishes in group (c), below werethen pretreated for 15 min in basal medium with a KDR-binding peptide,and then the cells in the dishes in groups (a), (b), and (c) were placedin fresh basal medium containing:

(a) no additives (negative control),

(b) 5 ng/mL VEGF (positive control), or

(c) 5 ng/mL VEGF plus the putative competing/inhibiting peptide.

After 5 min of treatment, lysates were prepared from each set of dishes.KDR was immunoprecipitated from the lysates was analyzed sequentially byimmunoblotting for phosphorylation with an anti-phosphotyrosineantibody, and for total KDR with an anti-KDR antibody (to control forsample loading).Results

As shown in FIG. 16, D1 was able to completely block the VEGF-inducedphosphorylation of KDR in HUVECs at 10 nM. More than half of thephosphorylation was inhibited by the compound at 1 nM. Homodimers D2 andD3, made up of the two individual binding moieties that are contained inD1, had no effect on phosphorylation at up to 100 nM, demonstrating thebenefit of heterodimer constructs in blocking a receptor-ligandinteraction. In multiple experiments, the IC₅₀ for D1 in this assayvaried between 0.5 and 1 nM. A different heterodimer containingunrelated binding sequences, D28, a tail-to-tail heterodimer comprisingthe polypeptides of SEQ ID NO: 305 and SEQ ID NO: 306 (FIG. 61), had noeffect on phosphorylation at 100 nM in spite of it's high bindingaffinity (11 nM for KDR by SPR), suggesting that the choice ofKDR-binding moieties is important when constructing a multimer tocompete with VEGF for binding to KDR. One of ordinary skill in the artwould be able to construct suitable heteromultimers using the bindingpolypeptides provided herein and routine screening assays.

Even though the affinity of D1 for KDR is 10-fold higher than that of D2(by SPR analysis), the IC₅₀ of D1 in the activation assay is at least100-fold lower. This suggests that targeting two distinct epitopes onKDR with a single binding molecule can generate greater steric hindrancethan a molecule with similar affinity that only binds to a singleepitope on KDR and, therefore, improve the ability to inhibit VEGFinduced KDR activity. Similarly, it should be pointed out that the twoKDR-binding moieties within D1, when tested as monomeric free peptides(SEQ ID NO: 277 and SEQ ID NO: 337 in the receptor activation assay, hadIC₅₀s of 0.1 and 1 micromolar, respectively. The IC₅₀ for the monomericfree peptides were 100 to 1000-fold higher than the IC₅₀ for D1 in theassay and 14 to 30-fold higher than the K_(D)s for the fluoresceinatedderivatives of the monomeric peptides. Thus, creating a dimer containingtwo peptides with weak VEGF-blocking activity has resulted in a moleculewith very potent VEGF-blocking activity that goes well beyond theincreased binding affinity of D1.

Example 15 Migration Assay

The following experiment assessed the ability of D1 to block theVEGF-induced migration of HUVECs in culture.

Protocol

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD Matrigel-coated FluoroBlok 24-well insert plates(#354141). Basal medium, containing either nothing or differentattractants such as VEGF (10 ng/mL) or serum (5% FBS) in the presence orabsence of potential VEGF-blocking/inhibiting compounds, was added tothe lower chamber of the wells. After 22 hours, quantitation of cellmigration/invasion was achieved by post-labeling cells in the insertplates with a fluorescent dye and measuring the fluorescence of theinvading/migrating cells in a fluorescent plate reader. The VEGF-inducedmigration was calculated by subtracting the migration that occurred whenonly basal medium was placed in the lower chamber of the wells.

Results

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by D1. At 5 nM D1, the VEGF-stimulatedendothelial cell migration was 84% blocked (see FIG. 17). At 25 nM D1,this migration was almost completely blocked. In other experiments, aknown KDR inhibitor, SU-1498 (Strawn, L. et al., 1996, Cancer Res.,56:3540-3545) was tested in the assay. SU-1498 at 3 micromolar did notblock the VEGF-induced migration as well as D1 (47% blocked at 3micromolar). D6 (structure shown below in Example 18), at 50 nM, alsoproduced essentially complete inhibition of the migration stimulated byVEGF. Serum was a very powerful attractant in the assay when used inplace of VEGF, but its effect was not significantly diminished by D1,indicating that D1 specifically inhibits endothelial migration inducedby VEGF.

Example 16 Preparation of Labeled Compounds

The following experiments describe methods used to prepare Tc, In, andI-labeled compounds.

Preparation of ^(99m)Tc-378(Ac-AGPTWC*EDDWYYC*WLFGTGGGK(PnAO₆—NH—(O═)C(CH₂)₃C(═O)—B)—NH₂; SEQ IDNO: 378)

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA*2.5 H₂O (Fluka) in 1 mL of water. The pHof the stannous DTPA solution was adjusted to pH 6-8 using 1N NaOH. SEQID NO: 378 (50 μg in 50 μL of 10% DMF) was mixed with 20 μL of^(99m)TcO₄ ⁻ (2.4 to 4 mCi, Syncor), followed by 100 μL of the stannousSn-DTPA solution. After 30 minutes at RT, the radiochemical purity (RCP)was 93%. The product was purified on a Supelco Discovery C16 amidecolumn (4×250 mm, 5 um pore size) eluted at a flow rate of 0.5 mL/minusing an aqueous/organic gradient of 1 g/L ammonium acetate in water (A)and acetonitrile (B). The following gradient was used: 30.5% B to 35% Bin 30 minutes, ramp up to 70% B in 10 min. The compound, which eluted ata retention time of 21.2 minutes was collected into 500 μL of 50 mMcitrate buffer (pH 5.2) containing 1% ascorbic acid and 0.1% HSA, andacetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of >98%.

Preparation of ¹¹¹In-Ac-AGPTWCEDDWYYCWLFGTJK(B-DOTA)-NH₂ (SEQ ID NO:338)

SEQ ID NO: 338 (50 μg in 50 μL of 10% DMF) was mixed with ¹¹¹InCl₃ (50μL, 400 μCi, Mallinckrodt) and 100 μL of 0.2M ammonium acetate orcitrate buffer at a pH of 5.3. After being heated at 85° C. for 45minutes, the radiochemical purity (RCP) ranged from 44% to 52.2% asdetermined using HPLC. The ¹¹¹In-labeled compound was separated fromunlabeled ligand using a Vydac C18 column (4.6×25 cm, 5 micron poresize) under following conditions: aqueous phase, 1 g/L ammonium acetate(pH 6.8); organic phase, acetonitrile. Gradient: 23% org. to 25% org. in30 minutes, up to 30% org. in 2 minutes, hold for 10 minutes. Thecompound, which eluted at a retention time of 20.8 min, was collectedinto 200 μL of 50 mM citrate buffer (pH 5.2) containing 1% ascorbic acidand 0.1% HSA, and the acetonitrile was removed using a Speed Vacuum(Savant). After purification the compound had an RCP of >93%.

Preparation of ¹¹¹In-D4

A histidine buffer was prepared by adjusting a 0.1M solution ofhistidine (Sigma) to pH 6.25 with concentrated ammonium hydroxide.Ammonium acetate buffer was prepared by adjusting a 0.2 M solution ofammonium acetate (99.99%, Aldrich) to pH 5.5 using concentrated HCl (J.T. Baker, Ultra Pure). High purity ¹¹¹InCl₃ (100 μL, 1.2 mCi,Malinckrodt, Hazelwood, Mo.) was added to D4 (200 μg in 200 of 50% DMF,10% DMSO, 20% acetonitrile and 20% water), followed by addition of 300μL of histidine buffer. The final pH was 5.5. After incubation of thereaction mixture at 85° C. for 45 minutes, the RCP was 20%.

Alternatively, ¹¹¹InCl₃ provided with a commercially availableOctreoScan™ Kit (134 μL, 0.6 mCi, Mallinkrodt) was added to D4 (135 μg)in 162 μL of 0.2M ammonium acetate buffer. The final pH was 5.5. Afterincubation of the reaction mixture at 85° C. for 45 min. the RCP was20%.

Preparation of ¹²⁵I-D5

D5 (200 μg), in 30 μL of DMF that had been previously adjusted to pH8.5-9.0 using diisopropyl amine, was added to 1 mCi of mono-iodinated¹²⁵I Bolton-Hunter Reagent (NEX-120, Perkin-Elmer) that had beenevaporated to dryness. The vial was shaken and then incubated on ice for30 minutes with occasional shaking After this time, the RCP was 23%.¹²⁵I-D5 was purified by HPLC at a flow rate of 1 mL/min using a VydacC18 column (4.6×250 mm, 5 micron pore size) under the followingconditions. Aqueous phase: 0.1% TFA in water; organic phase: 0.085% TFAin acetonitrile. Gradient: 30% org. to 36% org. in 30 minutes, up to 60%org. in 5 minutes, hold for 5 minutes. The compound was collected into200 μL of 50 mM citrate buffer (pH 5.2) containing 1% ascorbic acid and0.1% HSA. Acetonitrile was removed using Speed Vacuum (Savant). Theresulting compound had an RCP of 97% (see FIG. 65).

Preparation of ¹⁷⁷Lu-D11

D11 (5 μL of a ˜1 μg/μL solution in 0.05N NH₄OH/10% EtOH) was added to aglass insert microvial containing 80 μL of 0.2M NaOAc buffer, pH 5.6.Enough ¹⁷⁷Lu was added to bring the ligand:Lu ratio to ≦2:1 (1-5 mCi).The vial was crimp-sealed and heated at 100° C. for 15-20 minutes,cooled for 5 minutes, and treated with 3 μL of 1% Na₂EDTA.2H₂O in H₂O.The entire reaction mixture was injected onto a Supelco Discovery RPAmide C16 column (4 mm×250 mm×5 μm). The following HPLC conditions wereused: Column temperature=50° C., Solvent A=H₂O w/0.1% TFA, Solvent B=ACNw/0.085% TFA, gradient 0.6/0.25 mL/min A/B at t=0 minutes to 0.5/0.4mL/min A/B at t=60 minutes. The retention time for D11 was ˜40 minutes;that of ¹⁷⁷Lu-D11 was ˜42 minutes. The radioactive peak was collectedinto 0.7 mL of 0.05M citrate buffer, pH 5.3 containing 0.1% Human SerumAlbumin Fraction V and 1.0% Ascorbic Acid, and the mixture was spun downin a Savant Speed Vac to remove organic solvents. Radiochemical puritiesof greater than 80% were obtained.

Preparation of ¹⁷⁷Lu-D13

D13 (306 μg) was added to a 2-mL autosampler vial with a ˜450 μL conicalinsert and dissolved in 0.01N NH₄OH (50 μL). To this was added 300 μL of0.5M Ammonium Acetate containing Sodium Ascorbate, Sodium Gentisate,L-Methionine and L-Tryptophan each at 10 mg/mL, plus Human Serum AlbuminFraction V at 2 mg/mL, final pH=7.6 adjusted with NaOH. A 6.8 μL aliquotof ¹⁷⁷LuCl₃ in 0.05N HCl (39.3 mCi) was added, the vial wascrimp-sealed, warmed for 15 min at 37 C, cooled for ˜5 minutes, and 10μL, of 1% Na₂EDTA 2H₂O in H₂O was added. A 350 μL, aliquot of thereaction mixture was injected onto a Supelco Discovery RP Amide C16column (4 mm×250 mm×5 μm). The following HPLC conditions were used:column temperature=37 C, Solvent A=H₂O containing 2 g/L NH₄OAc buffer,pH 7.0, Solvent B=80% ACN/20% H₂O, gradient 0.56/0.24 mL/min A/B at t=0minutes to 0.47/0.33 mL/min A/B at t=30 minutes. The retention time forD13 was ˜28 minutes; the retention time for ¹⁷⁷Lu-BRU 1339 was ˜29minutes. The radioactive peak was collected into 1 mL of a buffercontaining Sodium Ascorbate, Sodium Gentisate, L-Methionine andL-Tryptophan each at 10 mg/mL, plus Human Serum Albumin Fraction V at 2mg/mL, final pH=7.6 adjusted with NaOH). It was then spun down ˜40minutes using a Speed Vacuum (Savant) to remove ACN. The RCP of theisolated product was 86%.

Preparation of ^(99m)Tc-D10

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA*2.5 H₂O (Fluka) in 1 mL of water. D10(100 μg in 100 μL of 50% DMF) was mixed with 75 μL of 0.1 M, pH 9phosphate buffer and 50 μL of ^(99m)TcO₄ ⁻ (2.4 to 5 mCi, Syncor),followed by 100 μL of the stannous Sn-DTPA solution. After 15 min at RT,the radiochemical purity (RCP) was 72%. The product was purified on aSupelco Discovery C16 amide column (4×250 mm, 5 um pore size) eluted ata flow rate of 0.7 mL/min using an aqueous/organic gradient of 0.1% TFAin water (A) and 0.085% TFA in acetonitrile (B; “ACN”). The followinggradient was used: 30% B to 42% B in 36 min, ramp up to 70% B in 10 min.The compound, which eluted at a retention time of 32 min., was collectedinto 500 μL of 50 mM citrate buffer (pH 5.2) containing 0.2% HSA, andacetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of >90%.

Preparation of ^(99m)Tc-D12

SnCl₂.2H2O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA*2.5 H₂O (Fluka) in 1 mL of water. D12(100 μg in 100 μL of 50% DMF) was mixed with 75 μL, of 0.1 M, pH 9phosphate buffer and 60 μL of ^(99m)TcO₄ ⁻ (2.4 to 4 mCi, Syncor),followed by 100 μL of the stannous Sn-DTPA solution. After 10 min at 40°C., the radiochemical purity (RCP) was 16%. The product was purified ona Supelco Discovery C16 amide column (4×250 mm, 5 um pore size) elutedat a flow rate of 0.7 mL/min using an aqueous/organic gradient of 0.1%TFA in water (A) and 0.085% TFA in acetonitrile (B). The followinggradient was used: 30% B to 42% B in 36 min, ramp up to 70% B in 10 min.The compound, which eluted at a retention time of 37.1 min. wascollected into 500 μL of 50 mM citrate buffer (pH 5.2) containing 0.2%HSA, and acetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of >90%.

Preparation of ^(99m)Tc-D14

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA*2.5 H₂O (Fluka) in 1 mL of water. D14(100 μg in 100 μL of 50% DMF) was mixed with 50 μL of ^(99m)TcO₄ ⁻ (6mCi, Syncor) and 125 μL of 0.1M phosphate buffer, pH 9 followed by 100μL of the stannous Sn-DTPA solution. After 15 min at 40° C., theradiochemical purity (RCP) was 21%. The product was purified on a Vydacpeptide C18 column (4.6×250 mm) eluted at a flow rate of 1 mL/min usingan aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA inacetonitrile (B). The following gradient was used: 30% B to 45% B in 40min. The compound, which eluted at a retention time of 34.9 min., wascollected into 500 μL of 50 mM citrate buffer (pH 5.3) containing 0.2%HSA, and acetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of 92.5%.

Preparation of ^(99m)Tc-D18

SnCl₂.2H2O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂ DTPA 2.5 H₂O (Fluka) in 1 mL of water. D18(100 μg in 100 μL of 50% DMF) was mixed with 50 μL of 0.1 M, pH 9phosphate buffer and 90 μL of ^(99m)TcO₄ ⁻ (14 mCi, Syncor), followed by100 μL of the stannous Sn-DTPA solution. The reaction was warmed for 20minutes at 37 C. The entire reaction was injected on a Vydac 218TP54 C18column (4.6×250 mm, 5 um silica) and eluted at a flow rate of 1.5 mL/minusing an aqueous/organic gradient of 0.1% TFA in water (A) and 0.085%TFA in ACN (B). The following gradient was used: 32% to 39% B in 30minutes, ramp up to 80% B in 2 min. The free ligand eluted at aretention time of 19 minutes. The complex, which eluted at 24 minutes,was collected into 500 μL of 50 mM citrate buffer (pH 5.3) containing0.1% HSA and 1% Ascorbic Acid. ACN and excess TFA were removed using aSpeed Vacuum (Savant) for 40 minutes. After purification, the compoundhad an RCP of 93%.

Preparation of ^(99m)Tc-D30

SnCl₂.2H₂O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of thissolution was added to 1 mL of a DTPA solution that was prepared bydissolving 10 mg of Ca Na₂DTPA*2.5 H₂O (Fluka) in 1 mL of water. D30(100 μg in 100 μL of DMF) was mixed with 150 μL of 0.1 M pH 8 phosphatebuffer and 50 μL of ^(99m)TcO₄ ⁻ (5.2 mCi, Syncor), followed by 100 μLof the stannous Sn-DTPA solution. After 15 min at 100° C., theradiochemical purity (RCP) was 13%. The product was purified on a VydacC18 peptide column (4.6×250 mm, 5 um pore size) eluted at a flow rate of1 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and0.085% TFA in acetonitrile (B). The following gradient was used: 10% Bto 50% B in 30 min, hold 50% B for 5 min, back to 70% B in 5 min. Thecompound, which eluted at a retention time of 33.2 min., was collectedinto 3 mL of 50 mM citrate buffer (pH 5.5) containing 0.2% HSA, andacetonitrile was removed using a Speed Vacuum (Savant). Afterpurification, the compound had an RCP of 92.4%.

Example 17 Binding to KDR-Transfected Cells

An experiment was performed to test the ability of ¹²⁵I-labeled D5 tobind to KDR-transfected 293H cells. In this experiment, differentamounts of ¹²⁵I-labeled D5 (1-4 μCi/mL, labeled with ¹²⁵I-Bolton-Hunterreagent and HPLC-purified) were incubated with mock and KDR-transfected293H cells in 96-well plates for 1 hr at room temperature. Binding wasperformed with and without 40% mouse serum to evaluate the serum effecton binding to KDR-transfected cells. After washing away the unboundcompound, the cells in each well were lysed with 0.5 N NaOH and thelysates were counted with a gamma counter.

The results of this experiment are summarized in FIG. 18 and FIG. 19.¹²⁵I-labeled D5 is able to specifically bind to KDR-transfected cells,and its binding is not affected by the presence of 40% mouse serum.Somewhat more binding to KDR-transfected cells was observed in theabsence of serum as compared to binding in the presence of 40% mouseserum. However, the binding of ¹²⁵I-D5 to mock-transfected cells wasalso increased by about the same extent when serum was omitted duringthe assay, indicating that the increased binding in the absence of serumwas non-specific (FIG. 18). Specific binding to KDR-transfected cells(after subtracting binding to mock-transfected cells) looked almostidentical with or without mouse serum (as shown in FIG. 19). In thisexperiment, 10-14% of the total CPM added were specifically bound toKDR-transfected cells (data not shown).

Example 18 Biacore Analysis of Heterodimer Binding to KDR-Fc andDetermination of Affinity Constant

A peptide heterodimer (FIG. 63) composed of SEQ ID NO: 277 and SEQ IDNO: 294 was prepared as previously described in Example 12 usingglutaric acid bis N-hydroxysuccinimidyl ester. The heterodimer wastested for binding to KDR-Fc using Biacore, and an affinity constant wasdetermined as follows.

Three densities of KDR-Fc were cross-linked to the dextran surface of aCM5 sensor chip by the standard amine coupling procedure (0.5 mg/mLsolution diluted 1:100 or 1:50 with 50 mM acetate, pH 6.0). Flow cell 1was activated and then blocked to serve as a reference subtraction.Final immobilization levels achieved:

R_(L) Fc 2 KDR-Fc=1607

R_(L) Fc 3 KDR-Fc=3001

R_(L) Fc 4 KDR-Fc=6319

Experiments were performed in PBS (5.5 mM phosphate, pH 7.65, 0.15 MNaCl)+0.005% P-20 (v/v)). D6 was diluted to 250 nM in PBS and serialdilutions were performed to produce 125, 62.5, 31.3 15.6, 7.8, and 3.9nM solutions. All samples were injected in duplicate. For association,peptides were injected at 20 μL/min for 12.5 minutes using the kinjectprogram. Following a 10 minute dissociation, any remaining peptide wasstripped from the KDR surface with a quickinject of 50 mM NaOH+1 M NaClfor 12 s at 75 μL/min. Sensorgrams were analyzed using BIAevaluationsoftware 3.1 and a hyperbolic double rectangular regression equation inSigmaPlot 6.0. Heterodimer steady state binding affinities (K_(DAV))were determined at all three KDR immobilization densities (Table 14).

TABLE 14 Summary of Parameters K_(D1) (nM) R_(max1) K_(DAV) (nM)R_(maxAV) R²* D6 Vs. 1600RU 46 13.1 1.5 12.6 0.995 Vs. 3000RU 25.5 21.20.665 22.7 0.991 Vs. 6000RU 17 61.3 0.662 62.2 0.993From these data, it appears that at the higher immobilization densities,the heterodimer binds KDR with a sub-nanomolar affinity (˜0.6 nM).

To assess the in vivo clearance of this peptide heterodimer, a smallamount of material was iodinated using iodogen and Na¹²⁵I according tostandard protocols (Pierce). One tube coated with the iodogen reagentwas pre-wet with 1 mL of 25 mM Tris, 0.4M NaCl, pH 7.5. This wasdiscarded and 100 μL of the same buffer added. Using a Hamilton syringe11 μL of the ¹²⁵I-NaI was transferred to the reaction tube. Based onoriginal estimates of the Na¹²⁵I concentration of 143.555 mCi/mt, the 11μL should contain about 1.5 mCi. After addition, the sample was swirledand set in a lead pig to incubate for 6 min with a swirl every 30 sec.After 6 min, the entire sample was transferred to the protein that wasin an Eppendorf tube. The sample was swirled and set to incubate for 8min, with a swirl every 30 sec. After 8 min the reaction was quenched(terminated) with tyrosine (10 mg/mL, a saturated solution), allowed tosit for 5 min, and then 2 μL was removed for a standard.

For purification a 10 mL column of the D-salt polyacrylamide 1800 wasused to separate the labeled peptide from labeled tyrosine. The columnwas first washed with 10 mL saline, then 5 mL of 25 mM Tris, 0.4M NaCl,pH 7.5 containing 2.5% HSA to block non-specific sites. After the HSAbuffer wash, the column was eluted with 60 mL of the 25 mM Tris, 0.4 MNaCl buffer, and the column was stored overnight at 4° C. The labeledsample contained 1.355 mCi, as determined by the dose calibrator. The 2μL sample that was removed as a standard contained 8.8 μCi. The peptidesample was applied to the D-salt 1800 column and eluted with theTris/NaCl buffer, pH 7.5. The flow was controlled by applying single 0.5mL aliquots for each fraction, #1-14, and then 1.0 mL for fractions25-43. FIG. 21 shows the elution profile of activity versus fractionnumber. The peak of activity in fractions #9, 10, and 11, was assumed tobe the peptide. The radioactivity in 24 through ˜40 is likely thelabeled tyrosine. From this purification, fractions #9-12 were pooledtogether and used for the subsequent clearance study (concentration of¹²⁵I-D6 in pool is 7.023 μg/mL; 100 μL=0.702 μg with 8.6 μCi).

A total of 15 mice were injected with 100 μL ¹²⁵I-D6 and mice (in setsof 3) were sacrificed at the following time points: 0, 7, 15, 30, 90minutes. After injection more than 2 μCi was found remaining in thesyringe, so actual activity injected was about 6 μCi. With 6 μCiinjected, the corresponding protein administered was ˜0.5 μg per animal.Once sacrificed, the counts were determined in a 50 μL plasma samplefrom each animal. For each set of three animals at each time point, thecounts were averaged, converted to % injected dose/ml plasma (ID %/mL),and then plotted to assess the rate of clearance (FIG. 20). This datawas fit to either a 4 or 5 parameter equation to determine the biphasichalf life of this molecule. The 4 parameter fit resulted in a T_(1/2α)of 2.55 minutes and a T_(1/2β) of 64.66 minutes. The 5 parameter fitresulted in a T_(1/2α) of 2.13 minutes and a T_(1/2β) of 23.26 minutes.

Larger volumes of plasma were also taken from mice sacrificed at the 0,30, and 90 minute time points. These samples were injected onto aSuperdex peptide column (Pharmacia) coupled to a radioactivity detectorto assess the association of the peptide with serum proteins (FIG. 21).As shown, the labeled peptide does associate with higher MW proteins,which could explain its biphasic half life clearance behavior.

To help assess the potency of the peptide as an anti-angiogenesisinhibitor, D6 was tested in an endothelial cell proliferation assayusing HUVECs and BrdU detection. Briefly, freshly isolated HUVECs(between p3-6) were cultured in RPMI+10% FCS+1% antibiotics+1%L-glutamine+0.4% BBE (bovine brain extract) and seeded per well,5000-10000/well in 100 μL. The cells were allowed to recover for 24 hrsprior to use. Then the cells were washed with PBS twice and treated for48 hrs with anti-VEGF antibody (positive control) or peptides A, B and C(0.1 and 10 ug/mL) in RPMI+0.1% BSA+1% L-glutamine. The following 6variables were tested in 2 series (n=4):

Series I: w/o VEGF

Series II: w/VEGF (30 ng/mL)

-   1. Standard medium: RPMI+10% FCS+1% antibiotics+1% L-glutamine+0.4%    BBE-   2. Negative control 1: RPMI (true starvation)-   3. Negative control 2: RPMI+0.1% BSA+1% L-glutamine-   4. Positive control: anti-VEGF 10 μg/mL in RPMI+0.1% BSA+1%    L-glutamine-   5. 0.1 μg/mL KDR peptides in RPMI+0.1% BSA+1% L-glutamine-   6. 10 μg/mL KDR peptides in RPMI+0.1% BSA+1% L-glutamine    Protocol:-   1) cells are incubated for 48 hours under various conditions-   2) 10 μL BrdU dilution (1:100 in EBM) is added to each well at 24    hours-   3) incubate for another 24 hours (total 48 hrs)-   4) aspirate the culture medium-   5) add 100 μL FixDenat (Roche Applied Science, Indianapolis, Ind.)    to each well, incubate at room temperature for 30 min.-   6) Discard FixDenat solution-   7) 100 μL antibody-solution (PBS 1% BSA and anti-BrdU PO) added to    each well.-   8) incubate at RT for 90 minutes.-   9) wash 3 times with PBS, 200 μL/well, 5 min.-   10) add substrate solution (TMB), incubate for 10-30 minutes-   11) transfer all to a flexible plate-   12) stop the reaction by adding 2 M H₂SO₄, 25 μL/well-   13) read absorbance at 450 nm within 5 minutes after stopping the    reaction.

Background binding was determined by omitting the anti-BrdU antibody in4 wells with control cells (cultured in complete medium; EBM+BulletKit(Clonetics, BioWhittaker, Inc., MD) and by complete labeling of cellsthat was not exposed to BrdU.

Of the two KDR binding peptide tested (D6 and SEQ ID NO: 277) as shownin FIG. 22, D6 (A) completely inhibits HUVEC proliferation at 10 μg/mLin the presence of VEGF, similar to an anti-VEGF antibody (positivecontrol). On the other hand, SEQ ID NO: 277 (B, one of the peptides thatmake up the heterodimer) did not inhibit proliferation in this assay atthe highest concentration tested (10 μg/mL). As a result, theheterodimer shows an enhanced ability to compete with VEGF in comparisonwith SEQ ID NO: 277 alone.

Example 19 BIAcore Analysis—Murine KDR-Fc Binding of Peptide Dimers D1and D7

Using BIAcore, the binding constants of peptide dimers D1 (a heterodimerof SEQ ID NO: 277 and SEQ ID NO: 294 and D7 (a heterodimer of SEQ ID NO:264 and SEQ ID NO 294; see FIG. 67) for murine KDR-Fc were determined.

Procedure

Three densities of recombinant murine KDR-Fc were cross-linked to thedextran surface of a CM5 sensor chip by the standard amine couplingprocedure (0.5 mg/mL solution diluted 1:100 or 1:40 with 50 mM acetate,pH 6.0). Flow cell 1 was activated and then blocked to serve as areference subtraction. Final immobilization levels achieved:

R_(L) Fc 2 KDR-Fc=2770

R_(L) Fc 3 KDR-Fc=5085

R_(L) Fc 4 KDR-Fc=9265

Experiments were performed in PBS buffer (5.5 mM phosphate, pH 7.65,0.15 M NaCl)+0.005% P-20 (v/v)). SEQ ID NO: 277, run as a control, wasdiluted to 125 nM in PBS. Serial dilutions were performed to produce62.5, 31.3, 15.6, 7.8, and 3.9 nM solutions. D1 and D6 were diluted to50 nM in PBS and serial dilutions were performed to produce 25, 12.5,6.25, 3.13, 1.56, 0.78, and 0.39 nM solutions. All samples were injectedin duplicate. For association, peptides were injected at 30 μL/min for 3minutes using the kinject program. Following a 10 minute dissociation,any remaining peptide was stripped from the rmKDR-Fc surface with aquickinject of 50 mM NaOH+1 M NaCl for 12 s at 75 μL/min.

Sensorgrams were analyzed using the simultaneous k_(a)/k_(d) fittingprogram in the BIAevaluation software 3.1. The Results are shown inTable 15 and FIGS. 23-25. The fact that about the same K_(D)2 constantwas achieved for both heterodimers even when the density of receptor onthe sensor chip was reduced by half is consistent with multimericbinding of the heterodimers to individual receptors rather thancross-link-type binding between receptors.

TABLE 15 Summary of Kinetic Parameters ka1 (1/Ms) Kd1 (1/s) ka2 (1/RUs)kd2 (1/s) KD1^(#) (nM) KD2^(‡) (nM) Chi²* D1 vs. 2700RU 7.94E+05 0.01393.31E−04 5.96E−04 17.5 0.751 0.077 vs. 5000RU 5.54E+05 8.88E−03 1.17E−044.57E−04 16.0 0.825 0.323 D7 vs. 2700RU 7.59E+05 0.011 3.36E−04 6.44E−0414.5 0.848 0.082 vs. 5000RU 5.21E+05 7.39E−03 1.17E−04 4.68E−04 14.20.898 0.278 Fluorescein vs. 2700RU 1.02E+06 0.037 — — 36.4 — 0.073 SEQID NO: 277 vs. 5000RU 5.18E+05 0.0174 — — 33.6 — 0.167 ^(#)K_(D1) is acalculated K_(D) based on kd₁/ka₁ ^(‡)K_(D2) is a calculated K_(D) basedon kd₂/ka₁ (i.e., avidity factor) *The chi2 value is a standardstatistical measure of the closeness of the fit. For good fitting toideal data, chi2 is of the same order of magnitude as the instrumentnoise in RU (typically < 2).

Example 20 In Vivo Inhibition of Tumor Growth

Conditions are described providing methods for determining efficacy ofthree (3) concentrations for Test Article (binding peptide, D6)suspected of having anti-angiogenic activity on SW-480 human coloncarcinoma cells using an in vivo xenograft tumor model.

Athymic nude mice are acceptable hosts for the growth of allogenic andheterogenic cells. Nude mice are required in Points to Consider in theCharacterization of Cell Lines used to Produce Biologicals (Points toConsider in the Characterization of Cell Lines used to ProduceBiologicals, FDA 1993).

D6 is a synthetic heterodimeric peptide suspected of havinganti-angiogenic activity. This peptide binds to the human VEGF receptor2 (KDR) with high affinity and competes with VEGF binding.

SW-480 Human Carcinoma Cells

Colon carcinoma, SW-480, cells (ATCC) were cultured in Dulbecco'sModified Eagles Medium (DMEM) supplemented with 4 mM L-glutamine, 0.1 mMnon-essential amino acids, 50 mg/mL Gentamicin, 250 mg/mL Fungizone and10% heat inactivated fetal bovine serum at 37° C. in 95% air and 5% CO₂.

Exponentially growing cells were harvested, washed twice in phosphatebuffered saline (PBS) to remove any traces of trypsin or serum. Cellswere suspended in Hanks Balanced Salt Solution (HBSS) for injections.

Sterile phosphate buffered saline (BioWhittaker) was manufactured inaccordance with cGMP regulations and was cell culture tested to assurecompatibility; having a pH of 7.3-7.7 and an osmolarity of 271-287mOsm/kg. PBS was the vehicle used to reconstitute Test Articles and forvehicle control injections.

Cisplatin (American Pharmaceutical Partners, Inc.; Los Angeles, Calif.)was prepared according to manufacture's specifications. Cisplatin wasprepared in an aseptic fashion using a BL2 BioChem guard hood.

Test System

-   A. Species/Strain: Mus musculus, Crl:NU/NU-nuBR mice (nude mice)-   B. Sex: Female-   C. Age: 6-8 weeks at initiation of treatment-   D. Weight Range: No weight requirement-   E. Source: Animals were received from the Gnottobiotic Department at    Charles River Laboratories, Wilmington, Mass.-   F. Number: A total of 115 animals were received and injected for    this study, with 90 mice used on study.-   G. Method of Identification:

Mice were uniquely numbered using an ear tag system. Additionally, cageswere marked with cage cards minimally identifying group number, animalnumber, study number and IACUC protocol number.

-   H. Randomization:

Animals were randomly assigned to treatment groups using Microsoft®Excel 97 SR-1 program.

-   I. Humane Care of Animals:

Treatment and care of the animals were in accordance with the standardoperating procedures of Charles River Laboratories, which adheres to theregulations outlined in the USDA Animal Welfare Act (9 CFR, Parts 1, 2,and 3) and the Guide for the Care and Use of Laboratory Animals.

This study protocol was covered under the Charles River LaboratoriesInstitutional Animal Care and Use Committee (IACUC Protocol Number:P071820011).

Animal Care

A. Diet and Drinking Water:

Mice were fed gamma-irradiated rodent chow ad libitum. Tap water wassterilized and supplied via bottle and sipper tube ad libitum.

B. Animal Environment:

Animals were housed by groups in semi-rigid isolators. Mice were housedin flat bottom caging containing five to ten animals. Cages containedgamma-irradiated contact bedding. The number of mice in each cage mayhave been altered due to the behavior of the mice, changes were noted inthe isolator inventory. The housing conforms to the recommendations setforth in the Guide for the Care and Use of Laboratory Animals, NationalAcademy Press, Washington, D.C., 1996 and all subsequent revisions.

Environmental controls were set to maintain a temperature of 16-26° C.(70±8° F.) with a relative humidity of 30-70. A 12:12 hour light: darkcycle was maintained.

C. Acclimation:

Once animals were received, they were allowed to acclimate to thelaboratory environment for 24-hours prior to the study start. Mice wereobserved for signs of disease, unusual food and/or water consumption orother general signs of poor condition. At the time of animal receipt,animals were clinically observed and appeared to be healthy.

Experimental Design

A. General Description:

Female athymic nude mice (Crl:NU/NU-nuBR) at 6-8 weeks of age were usedin this study. A total of 115 mice were injected subcutaneously into theright lateral thorax with 5×10⁶ SW-480, human colon carcinoma cells.When tumors reached a target window size of approximately 150±75 mg, 90tumor-bearing mice were randomly selected and distributed into one ofnine groups. Test Articles and vehicle were administeredintraperitoneally (IP), Cisplatin was administered intravenously (IV).Tumor measurements were recorded twice weekly using hand-held calipers.Mice were monitored daily for signs of toxicity and morbidity. At studytermination, animals were euthanized by carbon dioxide overdose andnecropsied for tissue collection.

B. Group Assignments:

A total of nine (9) groups were used in this study. Each group containedten (10) tumor-bearing mice. Groups 1 and 2 contained untreated andvehicle treated negative control mice, respectively. Groups 3, 4, and 5contained mice that received one of three different concentrations ofthe D6 anti-angiogenic peptide. Group 9 contained mice that receivedcisplatin, a standard chemotherapeutic compound as a positive control.

C. Dosing Levels and Regiment:

Dose levels for each group are provided in Table 16. Dosing began thesame day that animals were randomly sorted into groups (Study Day 7).Each dose was removed from the dose vial using aseptic technique foreach animal and the injection site was wiped with an alcohol swab priorto dose administration. Doses were administered with a 1.0 mL syringeand a 27-gauge×½″ needle for each mouse

The Test Article- and vehicle-treated mice received dailyintraperitoneal (IP) injections for 15 days. Cisplatin was administeredevery other workday for a total of five (5) doses via an intravenousroute.

TABLE 16 Study Treatment Groups Test Concentration Number of GroupArticle mg/kg Animals 1 Untreated — 10 2 Vehicle 0 10 3 D6 0.05 10 4 D60.5 10 5 D6 5.0 10 9 Cisplatin 6.0 10D. Clinical Observations of Animals:

Clinical Observations of each animal were performed and recorded atleast once daily for toxicity, morbidity and mortality. Morbidityincluded signs of illness such as, but not limited to, emaciation,dehydration, lethargy, hunched posture, unkempt appearance, dyspnea andurine or fecal staining

E. Tumor Measurements:

In accordance with the protocol tumor measurements were taken twiceweekly throughout the study by measuring the length and width of tumorswith calibrated calipers. Measurements occurred a minimum of 3-4 daysapart, except when animals were euthanized and measurements were taken;this sometimes resulted in an interval of less than 3 days. Tumorweights were calculated using the following formula: mg=(L×W²)/2.Animals were euthanized either when mean tumor weight was ≧1000 mg pergroup over two (2) consecutive measurements, or if tumors becameulcerated, impaired the animal's ability to ambulate or obtain food andwater.

F. Unscheduled Euthanasia and Unexpected Deaths:

1. Unscheduled Euthanasia:

None of the animals required unscheduled euthanasia while on study.

2. Unexpected Deaths:

None of the animals died while on study.

G. Necropsy:

1. Euthanasia and Necropsy Order:

All mice in groups 1, 2, 3, 4, and 5 (50 total) were submitted fornecropsy when tumors reached a group mean target size of ≧1000 mg overtwo (2) consecutive measurements within a group. Animals were submittedfor necropsy to the Charles River Laboratories Health MonitoringLaboratory (HM), Wilmington, Mass. All animals were euthanized on StudyDay 22, short of received the full 28 day treatment regiment with TestArticles because mean tumor size was ≧1000 mg in Test Article TreatedGroups 3-8.

All animals were humanely euthanized by carbon dioxide (CO₂) inhalation.

2. Tissue Collection:

Tumors were dissected free of surrounding tissue and overlying skin.Additionally the kidneys were collected. Any abnormalities noted on therenal surfaces were noted.

Frozen blocks were made of tumors and kidneys for each animal. Arepresentative section of the tissue (tumor, kidneys) was taken. Kidneysections included the cortex and medulla. Tissue sections were placed inthe bottom of a labeled plastic-freezing mold. Tissue was embedded withOCT medium. Blocks were submerged into isopentane chilled with dry iceuntil frozen. Blocks were briefly examined for quality, and stored ondry ice.

Blocks were labeled with the animal number and a letter codecorresponding to tissue (A=left kidney; B=right kidney; C=mass). Blocksfrom one animal were placed into a labeled bag.

Results

A. In-Life Measurements and Observations

1. Clinical Observations, Morbidity and Mortality Summary Statement:

All animals appeared healthy and were within normal limits throughoutthe study. D6 showed no signs of toxicity at the doses used in thisstudy.

Animals were euthanized on Study Day 22. All mice, except Group 9 mice,were euthanized prior to completing Test Article administration, becausemean tumor size was ≧1000 mg in Groups 1-8. Group 9, Cisplatin-treatedanimals were euthanized on Study Day 22 when mean tumor weight was 995mg. No animals died while on study.

2. Mass Palpation Summary Statement:

Throughout the study palpable masses were detected in all mice, withtumors progressively growing for the duration of the study. As expectedtumors in untreated and vehicle treated negative control mice (Groups 1and 2) grew the fastest, reaching a mean tumor size of 1000 mg on orbefore Study Day 20. In addition, animals treated with Cisplatin (Group9) developed tumors that grew the slowest reaching a mean tumor size of995 mg at study termination (Day 22).

In general, except for Group 3 mice, all animals treated with TestArticle resulted in slower tumor growth (FIG. 65). Animals in Group 3,which were treated with the low dose of D6 (0.05 mg/kg) had tumors thatgrew at approximately the same rate as the tumors in untreated andvehicle treated animals in Groups 1 and 2. Animals treated with eitherhigher doses of D6 (Groups 4 and 5) had tumors that grew slower;reaching a mean tumor size of 1000 mg on Study Day 21. When compared tocontrol Groups 1 and 2 mice, Test Article treatment resulted in a delayof tumor growth of approximately 1 day.

B. Conclusions

Data from this study validate the model used because tumor-bearing micein negative control Groups 1 and 2 and positive control Group 9responded as expected.

Throughout the study palpable masses were observed in all groups. Inaddition, all animals were healthy and within normal limits throughoutthe study. Furthermore, D6 did not adversely affect the animals.Therefore, these data would suggest that animals treated with D6 TestArticle had tumors that grew slowly (approximately 1 day slower over the22 day test period than controls). Also, since the Test Article did notshow any significant toxic effects, higher concentrations of TestArticle could also be used with potentially better tumor regression.

TABLE 17 Days Test Article After Un- D6 Cell treated Vehicle 0.005 0.050.5 Cisplatnin Injection Control Control mg/kg mg/kg mg/kg 6 mg/kg TumorWeights (mg) 4 48 49 43 51 50 34 7 164 156 157 163 154 160 8 180 164 156133 168 173 11 340 388 333 298 310 407 14 684 648 726 596 577 675 201064 986 973 857 978 635 21 1412 1571 1468 983 1056 839 22 1967 18632026 1474 1526 995

Example 21 In Vitro Cell Proliferation Assay

Microvascular endothelial cells (MVECs, Cascade Biologics, Portland,Oreg.) were used to assess the in vitro efficacy of D6 and relatedanalogues for their ability to inhibit VEGF-stimulated proliferation.MVECs (passage 2) were grown to 90% confluency, trypsinized and platedin gelatin-coated 96-well microtiter plates at a density of 4−8×10³cells/well. Sixteen to 24 hours after plating, the cells were washed onetime (2004/well) with media devoid of fetal bovine serum but containing0.1% bovine serum albumin (BSA). Fresh BSA-containing media was added toeach well and the cells were incubated for an additional 24 hours. Afterthis 24 hour period of starvation, fresh BSA-containing media(containing 25 ng/mL VEGF) with or without D6 was added and the cellswere incubated for an additional 48 hours at 37 C. To assess the dosereponse in this assay, multiple D6 concentrations were tested induplicate wells. The media was removed and fresh BSA-containing mediawas added with or without BrdU and the cells were incubated for anadditional 24 hours prior to determining the level of incorporationexactly as described by the manufacturer. Results are shown in FIG. 84.

Example 22

The following experiment assessed the ability of D25 and D27 to blockthe VEGF-induced migration of HUVECs in culture and demonstrated thatthe added glycosylation and/or distinct spacer structure used in D27enhanced its potency.

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, with or without VEGF (10 ng/mL) in the presence or absenceof D25 or D27, was added to the lower chamber of the wells. After 22hours, quantitation of cell migration/invasion was achieved bypost-labeling cells in the insert plates with a fluorescent dye andmeasuring the fluorescence of the invading/migrating cells in afluorescent plate reader. The VEGF-induced migration was calculated foreach experimental condition by subtracting the amount of migration thatoccurred when only basal medium was added to the lower chamber of thewells.

Results:

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by both D25 and D27 (FIG. 66). D27 wasten-fold more potent than D25 (IC₅₀ 0.5 nM and 5 nM respectively),indicating that the glycosylation of D27 and/or its distinct spacerproperties has enhanced its ability to bind KDR and block the effects ofVEGF.

Example 23

The following experiment assessed the ability of “Adjunct A” multimericconstruct of TKPPR peptide (SEQ ID NO: 503; binds to NP-1, a VEGFreceptor that enhances the effects of VEGF mediated by KDR), to enhancethe inhibition of the VEGF-induced migration of HUVECs in cultureproduced by D6. AdjunctA=5CF-Gly-N{[CH₂CH₂C(═O)-Gly-N(CH₂CH₂C(═O)-Adoa-Thr-Lys-Pro-Pro-Arg-OH]₂}₂where Adoa=3,6-dioxa-8-aminooctanoyl, 5CF=5-carboxyfluoresceinyl. SeeFIG. 67B.

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, containing with or without VEGF (10 ng/mL) in the presenceor absence of varying concentrations of D6, or varying concentrations ofD6 in combination with a constant 100 nM Adjunct A (synthesized asdescribed in WO 01/91805 A2), was added to the lower chamber of thewells. After 22 hours, quantitation of cell migration/invasion wasachieved by post-labeling cells in the insert plates with a fluorescentdye and measuring the fluorescence of the invading/migrating cells in afluorescent plate reader. VEGF-induced migration was calculated for eachexperimental condition by subtracting the amount of migration observedin the absence of VEGF.

Results:

VEGF induced a large increase in endothelial cell migration in theassay, which was potently blocked by D6 (IC₅₀ about 12.5 nM), but not by100 nM Adjunct A alone (FIG. 67A). Surprisingly however, Adjunct Awasable to enhance the potency of D6 by about ten-fold when used in theassay simultaneously with D6 (IC₅₀ about 2.5 nM). This indicates thatcompounds containing the TKPPR sequence (or similar) found in Adjunct Acan be used to enhance the potency of certain compounds such as D6,which compete with VEGF for binding to KDR. In addition, aheteromultimer containing the peptide sequences found in D6 or similar)as well as the TKPPR sequence (or similar), in one or more repetitions,would likely possess enhanced activity in this assay. See U.S. patentapplication Ser. No. 09/871,974, incorporated by reference in itsentirety, for details regarding the preparation of TKPPR constructs.

Example 24 Synthesis of D27

Synthesis of 1 and 3 (see FIGS. 68 and 69)

Synthesis of the monomers were carried out as described in Method 5 on a0.25 mmol scale employing as the starting resinFmoc-GGGK(iV-Dde)NH-PAL-PEG-PS resin. The peptide resin was washed anddried before cleavage or further derivatization by automated or manualmethods.

Procedure Synthesis of Peptide 2 and Peptide 4 (see FIGS. 68 and 69)

Appendage of Biotin-JJ, Lysyl, Glycyl and Serinyl (GalNAc(Ac)₃-α-Dmoieties onto 1 and 3 was done by manual SPPS such as described inMethod 6 and Method 8. The coupling of amino acids was carried out inDMF using HOBt/DIC activation (except for Ser(GalNAc(Ac)₃-α-D). Fmocremoval was carried out with 20% piperidine in DMF. All couplings were5-16 hours duration. After each coupling, the completion was confirmedby the Kaiser test. In the case of Ser(GalNAc(Ac)₃-α-D, the coupling wasperformed in DMF employing HATU/DIEA as the coupling agent. In caseswhere the Kaiser test indicated unreacted amino groups the coupling wasrepeated. Removal of the N-terminal Fmoc group and cleavage from resinwas performed. The crude peptide was precipitated in ether and washedtwice by ether and dried under vacuum. The linear crude peptide wasdirectly cyclized by dissolving the peptide in DMSO (40 mg/mL). The pHof the solyution was adjusted to 8 by addition of aqueousN-methylglucamine. and the solution was stirred in air for 48 h at roomtemperature. The peptides were then purified employing gradient HPLC asdescribed in Method 1 employing a Waters-YMC C-18 ODS preparative column(250 mm×4.6 mm i.d.). The pure product-containing fractions werecombined and lyophilized to provide the needed peptides.

Procedure: Synthesis of D27—Compound 6 (see FIG. 70)

To a solution of glutaric acid bis-NHS ester (0.122 mmol, PierceScientific Co.) in anhydrous DMF was added dropwise a solution of 4 inDMF (40 mg, 0.0122 mmol, DIEA was added to neutralize thetrifluoroacetic acid bound to the peptide and N-hydroxysuccinimideformed during the reaction). This 0.7 mL solution was stirred for 4 h.The reaction was monitored by HPLC and mass spectroscopy. DMF wasremoved under vacuum. The excess diester was removed by addition ofethyl acetate, which precipitated the peptide-monoester 5 whiledissolving glutaric acid bis-NHS ester. The mixture was centrifuged andthe liquid portion decanted. This was repeated twice. The residue waskept under vacuum for 10 min. The residue was dissolved in DMF and mixedwith a solution of 2 (37 mg, 0.009 mmol) in DMF (pH 7). It was stirredat ambient temperature for 16 h. The volatiles were removed under highvacuum and the acetate functions were removed by treatment of theresidue with 1 mL of hydrazine/MeOH (15/85, v/v) solution with stirringfor 2.5 h at ambient temperature. Acetone was added to quench the excessof hydrazine and the volatiles were removed under vacuum. The resultingresidue was dissolved in DMSO and purified by preparative HPLC asdescribed above to provide 9 mg of the pure material.

Sequence and Analytical Data for Peptides 2, 4 and 6

HPLC Compound Ret. time Mass Spectrum identifier Sequence (System)(ESI, neg. ion) Peptide 2: Ac- 7.4 min 2041.3 [M − 2H]/2 New Seq, aAGPTWCEDDWYYCWLFGTGGGK{Biotin- (T) Seq 11JJK[NH₂-Ser(GalNAc(Ac)₃-α-D)-Gly- derivative Ser(GalNAc(Ac)₃-α-D]}—NH₂Peptide 4: Ac-VCWEDSWGGEVCFRYDPGGGK(NH₂ 8.0 min 1636.3 [M − 2H]/2New Seq, a Ser(GalNAc(Ac)₃-α-D)-Gly- (T) Seq 5 Ser(GalNAc(Ac)₃-α-D)—NH₂derivative D27 Ac-AGPTWCEDDWYYCWLFGTGGGGK{Ac- 5.50 min  1737.2 (M − 4H)/VCWEDSWGGEVCFRYDPGGGK[S(GalNAc-α-D)- (M) 4; 1389.3G-S(GalNAc-α-D)-Glut-S(GalNAc-α-D)-G-S(GalNAc- (M − 5H)/5;α-D)—NH(CH₂)₄—(S)—CH(Biotin-JJNH—)C(═O)—]—NH₂}—NH₂ 1157.7 [M − 6H]/6System T: Column: Waters XTerra, 4.6×50 mm; Eluents: A: Water (0.1%TFA), B: Acetonitrile (0.1% TFA): Elution: Initial condition, 15% B,Linear Gradient 15-50% B in 8 min; Flow rate: 3.0 mL/min; Detection: UV@ 220 nm.

Example 25 Demonstration of the Distinction Between Binding Affinity andBiological Potency Through In Vitro Assays

The following experiments showed that heteromultimeric peptides candisplay much greater biological potency than a monomeric peptide withsimilar binding affinity to the same target.

Protocol Experiment 1:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described in Example 5. The cells were incubatedwith ¹²⁵I-VEGF in the presence or absence of SEQ ID NO: 504 or D1 (at300, 30, 3, and 0.3 nM). After washing the cells, the boundradioactivity was quantitated on a gamma counter. The percentageinhibition of VEGF binding was calculated using the formula[(Y1−Y2)×100/Y1], where Y1 is specific binding to KDR-transfected 293Hcells in the absence peptides, and Y2 is specific binding toKDR-transfected 293H cells in the presence of peptide competitors.Specific binding to KDR-transfected 293H cells was calculated bysubtracting the binding to mock-transfected 293H cells from the bindingto KDR-transfected 293H cells.

Protocol Experiment 2:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, with or without VEGF (10 ng/mL) in the presence or absenceof increasing concentrations of SEQ ID NO: 504 or D1, was added to thelower chamber of the wells. After 22 hours, quantitation of cellmigration/invasion was achieved by post-labeling cells in the insertplates with a fluorescent dye and measuring the fluorescence of theinvading/migrating cells in a fluorescent plate reader. VEGF-stimulatedmigration was derived by subtracting the basal migration measured in theabsence of VEGF.

Results Experiment 1:

As shown in FIG. 71, SEQ ID NO: 504 AND D1 competed about equally wellwith ¹²⁵I-VEGF for binding to KDR-transfected cells, indicating thatthey possess comparable binding affinities as well as a comparableability to inhibit VEGF from binding to KDR.

Results Experiment 2:

Despite the fact that both SEQ ID NO: 504 and D1 potently block¹²⁵I-VEGF binding to KDR-expressing cells to the same degree (FIG. 72),the heterodimeric D1 was much more potent in blocking the biologicaleffects of VEGF as demonstrated in an endothelial cell migration assay(FIG. 72) than the monomeric SEQ ID NO: 504. At up to 62.5 nM, a peptidecomprising SEQ ID NO: 504 had no effect on VEGF-stimulated migrationwhereas D1 completely blocked VEGF-stimulated migration at 50 nM. Thesedata suggest that heteromultimeric binding can more effectively blockthe biological activity of a ligand than a monomer, even when themonomer has a comparable ability to inhibit ligand binding to itsreceptor.

Example 26 Identification of Fragments of SEQ ID NO: 356 with KDRBinding Activity

The following experiment showed that fragments of SEQ ID NO: 356 canmaintain significant KDR binding activity.

Protocol:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described in Example 6. Binding of theneutravidin-HRP complexes to the cells was carried out as in Example 6with a complex concentration of 5.5 nM in the presence of 0 to 250 nM or0 to 1000 nM of the following competing peptides: SEQ ID NOS: 356, 462,463, and 465. After determining the specific binding under eachexperimental condition, the IC₅₀ for each peptide was determined (wherepossible).

Results:

As shown in Table 18, SEQ ID NO: 462, composed of just theAsp-Trp-Tyr-Tyr (SEQ ID NO: 490) binding motif that is also shared withSEQ ID NO: 286 along with the non-targeted Gly-Gly-Gly-Lys (SEQ ID NO:262) sequence that was added to most monomeric peptides synthesizedbased on phage display data, was the smallest fragment able to blockpeptide/neutravidin-HRP complex binding with an IC₅₀ below onemicromolar. Surprisingly, a larger fragment comprising SEQ ID NO: 356,failed to significantly inhibit complex binding at one micromolar.However, when a solubilising motif, (Gly-Arg-Gly)₃ was added to thelatter peptide to make SEQ ID NO: 465, it was able to compete with thecomplex for binding with an IC₅₀ of 175 nM, confirming that certainfragments of SEQ ID NO: 356 containing the Asp-Trp-Tyr-Tyr (SEQ ID NO:490) motif retain KDR-binding activity.

TABLE 18 Fragments of SEQ ID NO: 356 in a displacement assay competingwith a complex composed of binding peptide and neutravidin- HRP forbinding to KDR-expressing cells. Fragment (SEQ ID NO) IC₅₀, nM 356 93462 850 463 >1000 465 175

Example 27 Cell Based Assay for Binding of KDR/VEGF Complex Binders

The ability of a KDR/VEGF complex-binding peptide to selectively bind tothe KDR/VEGF complex was demonstrated.

Reagent Preparation

The reagents for this assay were prepared as described in Example 5except where noted.

Preparation of Peptide-¹²⁵I-Neutravidin Solution

Biotinylated peptides SEQ ID NOS: 321, 320 and 323, and a biotinylatednon-binding control peptide were used to prepare 1.25 μM stock solutionsin 50% DMSO. A 33.33 nM stock solution of ¹²⁵I-neutravidin was purchasedfrom Amersham (Buckinghamshire, UK). A stock solution of 13.33 nM¹²⁵I-neutravidin/100 nM VEGF was prepared by mixing 850 mL of¹²⁵I-neutravidin with 22 μL of 10 μM VEGF and 1275 μL of M199 media.Another stock solution was prepared in the same manner, but lackingVEGF. To prepare 13.33 nM peptide-¹²⁵I-neutravidin complexsolutions±VEGF, 500 μL of the ¹²⁵I-neutravidin (with and without VEGF)stock solutions (prepared in last step) were mixed with 24 μL of 1.25 μMpeptide solution of SEQ ID NOS: 321, 320 and 323, or control peptide.The mixtures were incubated on a rotator at 4 C for 60 minutes, followedby addition of 50 μL, of soft release avidin-sepharose (50% slurry inddH₂0) to remove excess peptides and another incubation for 30 minuteson a rotator at 4 C. Finally, the soft release avidin-sepharose waspelleted by centrifuging at 12,000 rpm for 5 minutes at roomtemperature, and the resulting supernatants were used for the assays.

Binding of Peptide/Neutravidin HRP to KDR-Transfected Cells

Complexes of control peptide and the test peptides (SEQ ID NOS: 321, 320and 323) with ¹²⁵I-neutravidin in the presence or absence of VEGF(prepared as above) were tested for their ability to bind 293H cellsthat were transiently-transfected with KDR. The complex of SEQ ID NO:321 with ¹²⁵I-neutravidin specifically bound to KDR-transfected 293Hcells as compared to mock transfected cells in the presence of VEGF(FIG. 73), but not where VEGF was omitted (FIG. 74). SEQ ID NO: 321, wasalso the best KDR/VEGF complex binder among the peptides tested usingfluorescence polarization and SPR (BIAcore) assays (Table 9). Thisexample shows that peptide (SEQ ID NO: 321) can specifically bind to theKDR/VEGF complex present on the cell surface. This establishes a utilityfor the assay as useful for targeting the KDR/VEGF complex in vitro andin vivo for diagnostic or therapeutic purposes. Since the KDR/VEGFbinding peptide only detects the functional and active KDR receptor andnot all the KDR present on cell surface, it will be useful in detectingand/or treating active angiogenesis in tumors, metastasis, diabeticretinopathy, psoriasis, and arthropathies.

Example 28

This example provides more evidence that heterodimeric peptidestargeting two epitopes on KDR are superior to a homodimeric peptide thatbinds one of the two epitopes on the target molecule. The followingexperiment provides further evidence that heterodimeric constructs aresuperior to homodimeric peptides in their ability to block thebiological effects of VEGF.

Protocol:

Serum-starved HUVECs were placed, 100,000 cells per well, into the upperchambers of BD fibronectin-coated FluoroBlok 24-well insert plates.Basal medium, containing either nothing or VEGF in the presence orabsence of increasing concentrations of homodimericD8 or heterodimericD17, was added to the lower chamber of the wells. After 22 hours,quantitation of cell migration/invasion was achieved by post-labelingcells in the insert plates with a fluorescent dye and measuring thefluorescence of the invading/migrating cells in a fluorescent platereader.

Results:

As shown in FIG. 75, VEGF induced a large increase in endothelial cellmigration in the assay, which was potently blocked by D17 but not D8.D17 blocked VEGF-induced migration with an IC₅₀ of about 250 nM while D8had no significant effect on migration even at 800 nM. This is in spiteof the fact that D8 used the full targeting sequence found in SEQ ID NO:356 while D17 contained a truncated version of the SEQ ID NO: 356sequence (as seen in SEQ ID NO: 465) with a lower affinity for KDR (asdemonstrated in Example 26). Thus a heterodimer with the capability ofbinding two separate epitopes on KDR is more effective at blockingligand binding to KDR than a homodimer containing the same or even morepotent targeting sequences.

Example 29 Preparation of KDR-Binding Peptides in which the DisulfideBond has been Replaced

Disulfide bond substitution analogs of SEQ ID NO: 301, where the Cysresidues at position 6 and 13 are replaced by a pair of amino acids, onewith a carboxy-bearing side-chain (either Glu or Asp) and the other withan amino-bearing side chain [(Lys or Dpr (2,3-diaminopropanoic acid)]were prepared. The cycle, encompassing the same sequence positions asthose included in SEQ ID NO: 301 (made by formation of the disulfidebond) was made by condensation of the side-chain amino and side-chainacid moieties, resulting in a lactam ring that bridges the residues 6-13as does the disulfide bond of SEQ ID NO: 301.

Table 19 below displays some examples of the substitutions made for Cys⁶and Cys¹³ of SEQ ID NO: 301 in lactam analogs.

TABLE 19 Lactam Analogs of SEQ ID NO: 277 Difference in PositionPosition Ring Size vs Sequence 6 13 SEQ ID NO: 277 SEQ ID NO: 277 CysCys — (parent seq) 453 Glu Lys 4 454 Lys Glu 4 455 Dpr Asp 0 456 Asp Dpr0 457 Asp Lys 3Synthesis of Resin bound SEQ ID NO: 453

Synthesis of 1 was carried out using Method 5 on a 0.25 mmol scale. Thepeptide resin 1 was washed and dried for further derivatization manually(see FIG. 76).

Synthesis of 4 (SEQ ID NO: 453)

To 1 (240 mg, 0.06 mmol) was added NMM (N-methyl morpholine)/HOAc/DMF1/2/10 (v/v/v) (65 mL). Palladium tris-triphenylphosphine [Pd(PPh₃)₄,554.4 mg, 0.48 mmol] was added and the resin was shaken for 20 hshielded from light. The resin was filtered and washed with a solutionof sodium diethyldithiocarbamate (0.5 g)/DIEA (0.5 mL)/DMF (100 mL), andfinally with DMF (3×70 mL). This treatment served to expose only thecarboxy and amino groups of Glu6 and Lys13 that are required for thelactam forming reaction. The on-resin cyclization of 2 was carried outusing HATU (114 mg, 0.3 mmol), NMM (66 μL, 0.6 mmol) and DMF (10 mL) for3 h. The completion of the cyclization was monitored by Kaiser test. Thepeptide was cleaved from the peptide resin 3 using reagent B for 4 h.The resin was filtered and the filtrate was evaporated to a paste. Thecrude peptide was precipitated in ether and washed twice with ether. Thecyclic peptide was purified by preparative reverse phase linear gradientHPLC using a Waters-YMC C-18 column (250 mm×30 mm i.d.) with CH₃CN intoH₂O (both with 0.1% TFA) as the eluent. Lyophilization of theproduct-containing fractions afforded 8 mg of (SEQ ID NO: 453). SEQ IDNOS: 454, 455, 456 and 457 were prepared similarly.

Example 30 Replacement of a Disulfide Bridge while Retaining KDR-BindingActivity

The following experiment demonstrated that the lactam SEQ ID NO: 454replaced a chemically reactive disulfide bridge to maintain significantKDR binding activity.

Protocol:

293H cells were transfected with the KDR cDNA or mock-transfected bystandard techniques described in Example 5. Neutravidin-HRP complexeswere prepared as in Example 5. Binding of the neutravidin-HRP complexesto the cells was carried out as in Example 5 with a complexconcentration of 5.5 nM in the presence of 0 to 250 nM SEQ ID NO: 277 orSEQ ID NO: 454. After determining the specific binding under eachexperimental condition, the IC₅₀ for each peptide was determined.

Results:

As shown in Table 20, SEQ ID NO: 454, containing a lactam disulfidebridge replacement, was still able to compete withpeptide-neutravidin-HRP complexes for binding to KDR although someaffinity was lost (IC₅₀ 108 nM versus 13 nM).

Table 20: SEQ ID NO: 277 and SEQ ID NO: 454 (disulfide bridgereplacement analog) in a displacement assay competing with aneutravidin-HRP/binding peptide complex for binding to KDR-expressingcells.

Fragment (SEQ ID NO) IC₅₀, nM 277 13 454 108

Example 31 Use of the Neutravidin/Avidin HRP Assay with BiotinylatedPeptides Identified by Phage Display Allows Identification of PeptidesCapable of Binding to the Target Even where the Affinity of the Peptidesis Too Low for Other Assays

This example confirms that the neutravidin/HRP screening assay describedherein is an effective technique for screening peptides whose affinityas monomers is too low for use in conventional screening assays, suchas, for example, an ELISA.

Three different derivatives of SEQ ID NO: 482, which was identified byphage display as a peptide that binds to cMet, were prepared asdescribed in U.S. Patent Application No. 60/451,588 (incorporated hereinby reference in its entirety), filed on the same date as U.S. patentapplication Ser. No. 10/382,082, of which the present application is acontinuation-in-part.

These three peptides and a control peptide that does not bind to cMet,were tested as tetrameric complexes with neutravidin HRP for theirability to bind cMet-expressing MB-231 cells. All three tetramericcomplexes of cMet-binding peptides bound to the MB231 cells as comparedto control peptide.

Cell Culture:

MDA-MB231 cells were obtained from ATCC and grown as monolayer culturein their recommended media plus 1 mL/L pen/strep (InVitrogen, Carlsbad,Calif.). Cells were split the day before the assay, 35000 cells wereadded to each well of a 96 well plate. The rest of the experiment wasconducted as in Example 6, except as noted below.

Binding of Peptide/Neutravidin HRP to MDA-MB-231 Cells:

Complexes of control peptide, and SEQ ID NO: 482 derivatives with 0, 1or 2 J spacers with neutravidin-HRP were prepared as described above andtested for their ability to bind MDA-MB-231 cells. During thepeptide/neutravidin-HRP complex preparation, a 7.5-fold excess ofbiotinylated peptides over neutravidin-HRP was used to make sure thatall four biotin binding sites on neutravidin were occupied. Aftercomplex formation, the excess of free biotinylated peptides was removedusing soft release avidin-sepharose to avoid any competition betweenfree biotinylated peptides and neutravidin HRP-complexed biotinylatedpeptides. The experiment was performed at several differentconcentrations of peptide/neutravidin-HRP, from 0.28 nM to 33.33 nM, togenerate saturation binding curves for derivatives with no or one spacer(FIG. 77) and 0.28 to 16.65 nM to generate a saturation binding curvefor the derivative with two spacers (FIG. 77). In order to draw thesaturation binding curve, the background binding of the controlpeptide/neutravidin HRP complex was subtracted from the binding of thebinding derivative peptide/neutravidin-HRP complexes for eachconcentration tested. Therefore, absorbance on the Y-axis of FIG. 77 isdifferential absorbance (cMet-binding peptide minus control peptide) andnot the absolute absorbance. Analysis of the saturation binding data inFIG. 77 using Graph Pad Prism software (version 3.0) yielded a K_(D) of12.62 nM (+/−3.16) for the tetrameric derivative with two spacers, 155.4nM (+/−86.56) for the tetrameric derivative with one spacer and 123.8 nM(+/−37.71) for the tetrameric derivative without a spacer. These bindingconstants are, as expected, lower than that measured by FP for therelated monodentate peptide SEQ ID NO: 482 (880 nM).

Results:

As was the case where the binding target was KDR, the neutravidin-HRPassay with biotinylated peptides identified with phage display wasuseful for identifying peptides capable of binding to an immobilizedtarget even when the affinity of the monomeric binding sequence is toolow for an ELISA-type assay (with washing steps after binding) to workwell (see FIG. 77).

Example 32 Binding of Tc-Labeled Heterodimeric Polypeptides toKDR-Transfected 293H Cells

The ability of Tc-labeled D10 to bind KDR was assessed usingKDR-transfected 293H cells. The results show that Tc-labeled D10 bindssignificantly better to KDR transfected 293H cells than to mocktransfected 293H cells, and good binding was maintained in the presenceof 40% mouse serum. In addition, a derivative of Tc-labeled D10 with itsamino acid sequence scrambled, D18, was shown to possess no affinity forKDR-expressing cells, confirming the specificity of the D10 binding tothose cells.

Transfection of 293H Cells

293H cells were transfected using the protocol described in Example 5.Transfection was done in black/clear 96-well plates (Becton Dickinson,cat. #354640). The cells in one half of the plate (48 wells) weremock-transfected (without DNA) and the cells in the other half of theplate were transfected with KDR cDNA. The cells were 80-90% confluent atthe time of transfection and completely confluent the next day, at thetime of the assay (the assay was aborted if these conditions were notsatisfied).

Preparation of Opti-MEMI Media with 0.1% HSA

Opti-MEMI was obtained from InVitrogen (Carlsbad, Calif.) and humanserum albumin (HSA) was obtained from Sigma (St. Louis, Mo.). Opti-MEMImedia was prepared by adding 0.1% HSA, 0.1% w/v HSA to opti-MEMI,followed by stirring at room temperature for 20 minutes. The media wasfilter sterilized using 0.2 μM filter.

Preparation of Tc-Labeled Peptide Dilutions for the Assay

D10 and D18 were diluted in opti-MEMI with 0.1% HSA to provide solutionswith final concentrations of 1.25, 2.5, 5.0, and 10 μCi/mL of eachTc-labeled heterodimer. A second set of dilutions was also preparedusing a mixture of 40% mouse serum/60% opti-MEMI with 0.1% HSA as thediluent.

Assay to Detect the Binding of the Tc-Labeled Heterodimers

Cells were used 24 h after transfection, and to prepare the cells forthe assay, they were washed once with 100 μL of room temperatureopti-MEMI with 0.1% HSA. After washing, the opti-MEMI with 0.1% HSA wasremoved from the plate and replaced with 70 μL of 1.25, 2.5, 5.0, and 10μCi/mL of Tc-labeled D10 or D18 (prepared as above with both diluentsolutions). Each dilution was added to three separate wells of mock- andKDR-transfected cells. After incubating at room temperature for 1 h, theplates were washed 5 times with 100 μL of cold binding buffer (opti-MEMIwith 0.1% HSA). 100 μL of solubilizing solution (0.5 N NaOH) was addedto each well and the plates were incubated at 37 C for 10 minutes. Thesolubilizing solution in each well was mixed by pipeting up and down,and transferred to 1.2 mL tubes. Each well was washed once with 100 μLof solubilizing solution and the washes were added to the corresponding1.2 mL tube. Each 1.2 mL tube was then transferred to a 15.7 mm×100 cmtube to be counted in an LKB Gamma Counter.

Binding of Tc-Labeled Peptide to KDR Transfected Cells

The ability of Tc-labeled D10 and D18 to bind specifically to KDR wasdemonstrated using transiently transfected 293H cells. As shown in FIG.78, Tc-labeled D10 bound better to KDR transfected 293H cells, ascompared to mock-transfected (with a scrambled peptide) 293H cells inboth the presence and absence of 40% mouse serum, although there wassome inhibition in the presence of serum. The total specific binding ofthis Tc-labeled heterodimer to KDR-expressing cells was greater thanthat observed previously with a Tc-labeled monomeric peptide (Example10). Tc-labeled D18, the scrambled peptide, displayed no affinity foreither mock-transfected or KDR-transfected 293H cells (not shown),confirming the specificity of D10 binding.

Example 33 Binding of a Lu-Labeled Heterodimeric Polypeptide toKDR-Transfected 293H Cells

The ability of Lu-labeled D13 to bind KDR was assessed usingKDR-transfected 293H cells. The results show that Lu-labeled D13 bindsbetter to KDR transfected 293H cells than to mock transfected 293Hcells, and significant binding was maintained in the presence of 40%mouse serum.

Transfection of 293H Cells

293H cells were transfected using the protocol described in Example 5.Transfection was performed in black/clear 96-well plates (BectonDickinson, San Jose, Calif.). The cells in one half of the plate (48wells) were mock-transfected (without DNA) and the cells in the otherhalf of the plate were transfected with KDR cDNA. The cells were 80-90%confluent at the time of transfection and completely confluent the nextday, at the time of assay (the assay was aborted if these conditionswere not satisfied).

Preparation of Opti-MEMI Media with 0.1% HSA

Opti-MEMI was prepared as in Example 32.

Preparation of Lu-Labeled Peptide Dilutions for the Assay

A stock solutions of Lu-labeled D13 was diluted in opti-MEMI with 0.1%HSA to provide solutions with final concentrations of 1.25, 2.5, 5.0,and 10 μCi/mL of labeled heterodimer. A second set of dilutions was alsoprepared using a mixture of 40% mouse serum/60% opti-MEMI with 0.1% HSAas the diluent.

Assay to Detect the Binding of the Lu-Labeled Heterodimers

Detection of binding was measured as detailed in Example 32 except thatLu-labeled D13 was used in place of the Tc-labeled heterodimers.

Binding of Lu-Labeled Peptide to KDR Transfected Cells

The ability of Lu-labeled D13 to bind specifically to KDR wasdemonstrated using transiently-transfected 293H cells. As shown in FIG.95, Lu-labeled D13 bound significantly better to KDR transfected 293Hcells, as compared to mock-transfected 293H cells in both the presenceand absence of 40% mouse serum, although there was some bindinginhibition in the presence of serum.

Example 34 Radiotherapy with a Lu-Labeled Heterodimeric Peptide inTumor-Bearing Mice

In this example, the ability of Lu-labeled D13 to inhibit the growth ofPC3 cell tumors implanted in nude mice is demonstrated.

Animal Model

PC3 cells from ATCC, grown as recommended by the supplier, were injectedsubcutaneously between the shoulder blades of nude mice. When theirtumors reached 100-400 mm³, twelve mice were injected i.v. with 500microcuries of Lu-labeled D13 and their growth monitored for anadditional 18 days. Mice were sacrificed if they lost 20% or more oftheir body weight or their tumors exceeded 2000 mm³. Tumor growth in thetreated mice was compared with the average tumor growth in 37 untreatednude mice implanted with PC3 tumors.

Results

In 6 of the 12 treated mice in the study, the tumors experienced asignificant or complete growth delay (FIG. 80) relative to untreatedtumor mice, indicating that D13 was effective in slowing PC3 tumorgrowth under the conditions employed.

Example 35 Preparation of Ultrasound Contrast Agents Conjugated toKDR-Binding Peptides

Ultrasound contrast agents comprising phospholipid-stabilizedmicrobubbles conjugated to KDR-binding polypeptides of the inventionwere prepared as described below.

200 mg of DSPC (distearoylphosphatidylcholine), 275 mg of DPPG.Na(distearoylphosphatidylglycerol sodium salt), 25 mg of N-MPB-PE weresolubilized at 60 C in 50 mL of Hexan/isopropanol (42/8). The solventwas evaporated under vacuum, and then PEG-4000 (35.046 g) was added tothe lipids and the mixture was solubilized in 106.92 g of t-butylalcohol at 60 C, in a water bath. The solution was filled in vials with1.5 mL of solution. The samples were rapidly frozen at −45 C andlyophilized. The air in the headspace was replaced with a mixture ofC₄F₁₀/Air (50/50) and vials capped and crimped. The lyophilized sampleswere reconstituted with 10 mL saline solution (0.9% —NaCl) per vial.

Peptide Conjugation

Peptides, e.g., SEQ ID NO: 356, SEQ ID NO: 294 and SEQ ID NO: 480, wereconjugated to a preparation of microbubbles as above described,according to the following methodology.

The thioacetylated peptide (200 μg, SEQ ID NO: 356) was dissolved in 204DMSO and then diluted in 1 mL of Phosphate Buffer Saline (PBS). Thissolution was mixed to the N-MPB-functionalized microbubbles dispersed in18 mL of PBS-EDTA 10 mM, pH 7.5 and 2 mL of deacetylation solution (50mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) wasadded. The headspace was filled with C₄F₁₀/Air (35/65) and the mixturewas incubated for 2.5 hours at room temperature under gentle agitation(rotating wheel), in the dark. Conjugated bubbles were washed bycentrifugation.

Example 36 Preparation of Ultrasound Contrast Agents Conjugated to KDRBinding Peptides

Ultrasound contrast agents comprising phospholipid-stabilizedmicrobubbles conjugated to KDR-binding polypeptides of the inventionwere prepared as described below.

Distilled water (30 mL) containing 6 mg of dipalmitoylphosphatidylserine(DPPS, Genzyme), 24 mg of distearoylphosphatidylcholine (DSPC, Genzyme)and 3 g of mannitol was heated to 65 C in 15 minutes then cooled to roomtemperature. N-MPB-DPPE(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]Na salt—Avanti Polar Lipids) was added (5% molar—1.9 mg).This derivatized phospholipid was dispersed in the aqueous phase usingan ultrasonic bath (Branson 1210—3 minutes).

Perfluoroheptane (2.4 mL from Fluka) was emulsified in this aqueousphase using a high speed homogenizer (Polytron®, 10000 rpm, 1 minute).

The emulsion was washed once by centrifugation (200 g/10 min) thenresuspended in 30 mL of a 10% solution of mannitol in distilled water.The washed emulsion was frozen (−45 C, 5 minutes) then freeze dried(under 0.2 mBar, for 24 hours).

Atmospheric pressure was restored by introducing a mixture of C₄F₁₀ andair. The lyophilizate was dissolved in distilled water (30 mL).Microbubbles were washed once by centrifugation and redispersed in 10 mLof Phosphate Buffer Saline.

Peptide Conjugation

Thioacetylated peptide (200 μg, SEQ ID NO: 356) was dissolved in 204DMSO and then diluted in 1 mL of Phosphate Buffer Saline (PBS). Thissolution was mixed to 5 mL of the N-MPB-functionalized microbubbles. 0.6mL of deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 Mhydroxylamine.HCl, pH 7.5) was added and the suspensions were stirred byinversion for 2 h30.

Microbubbles were washed twice with a solution of maltose 5% andPluronic F68 0.05% in distilled water, by centrifugation (200 g/10minutes). The final volume was fixed to 5 mL.

Example 37 Preparation of Ultrasound Contrast Agents Conjugated to KDRBinding Peptides

Ultrasound contrast agents comprising microballoons conjugated toKDR-binding polypeptides of the invention were prepared as describedbelow.

Distilled water (30 mL) containing 40 mg ofdistearoylphosphatidylglycerol (DSPG, Genzyme) was heated to 65 C during15 minutes then cooled to 40 C.

DPPE-PEG2000-Maleimide (3.5 mg—Avanti Polar Lipids) and tripalmitine (60mg—Fluka) were dissolved in cyclohexane (0.6 mL) at 40 C in a ultrasoundbath for 2 min.

This organic phase was emulsified in the aqueous phase using a highspeed homogenizer (Polytron®, 10000 rpm, 1 minute).

Polyvinylalcohol (200 mg) dissolved in distilled water (5 mL) was addedto the emulsion. The mixture was cooled to 5 C, then frozen (−45 C, 10minutes) and finally freeze dried (under 0.2 mBar, for 24 hours).

The lyophilisate was dispersed in distilled water (15 mL). The mixturewas stirred for 30 min to obtain a homogenous suspension ofmicroballoons.

Peptide Conjugation

The thioacetylated peptide (200 μg) was dissolved in 204, DMSO thendiluted with PBS (1 mL).

7.5 mL of the suspension of microballoons obtained as above describedwere centrifuged (500 rpm for 5 min). The infranatant was discarded andmicroballoons were redispersed in Phosphate Buffer Saline (2 mL).

The microcapsule suspension was mixed with the solution of peptide.Three hundred microliters of a hydroxylamine solution (10.4 mg in PBS 50mM, pH: 7.5) was added to the suspension to deprotect the thiol. Thesuspension was stirred by inversion for two and a half hours.

The microballoons were washed twice by centrifugation (500 g/5 min) withdistilled water containing 5% maltose and 0.05% Pluronic F68 and finallyredispersed in 3 mL of this solution.

Example 38 Ultrasound Contrast Agents Conjugated to KDR BindingPolypeptides Bind to KDR-Expresing Cells In Vitro and In Vivo

The ability of ultrasound contrast agents conjugated to peptides of theinvention to bind to KDR-expressing cells in vitro was assessed using293H cells transfected to express KDR. Additionally, the ability ofultrasound contrast agents conjugated to KDR binding polypeptides of theinvention to bind to KDR-expressing tissue in vivo was assessed usingtwo known models of angiogenesis, the rat matrigel model and the ratMatB III tumor model.

Transfection of 293H Cells on Thermanox® Coverslips

293H cells were transfected with KDR DNA as set forth in Example 5. Thetransfected cells were incubated with a suspension of peptide-conjugatedultrasound contrast agents or with a control peptide (a scrambledversion of the conjugated peptide having no affinity for KDR).

For the incubation with the transfected cells a small plastic cap isfilled with a suspension containing 1 to 3×10⁸ peptide-conjugatedmicrobubbles and the cap covered with an inverted Thermanox® coverslipas to put the transfected cells in contact with the conjugatedmicrobubbles. After about 20 min at RT, the coverslip is lifted withtweezers, rinsed three times in PBS and examined under a microscope toassess binding of the conjugated microbubbles.

FIG. 85 indicates that microballoons conjugated to peptides of theinvention bind specifically to KDR-expressing cells. Indeed,microballoons conjugated to KDR-binding peptide bound to KDR-expressingcells while they did not bind appreciably to mock transfected cells andmicroballoons bearing a scrambled control peptide showed no appreciablebinding.

Determination of the % of Surface Covered by Microvesicles

Images were acquired with a digital camera DC300F (Leica) and thepercent of surface covered by bound microbubbles or microballoons in theimaged area was determined using the software QWin (Leica MicrosystemAG, Basel, Switzerland).

The following table shows the results of the binding affinity (expressedas coverage % of the imaged surface) of targeted microvesicles of theinvention to KDR transfected cells, as compared to the binding of thesame targeted microvesicles towards Mock-transfected cells or (only inthe case of the peptide) to the binding of microvesicles targeted with ascrambled peptide to the same KDR transfected cells.

As shown in Table 21, targeted microvesicles show increased bindingaffinity for KDR.

TABLE 21 Coverage % SEQ Scrambled ID NO KDR Mock peptide Example 35 35614.2% 1.4% 2.1% 277 3.5% 0.9% n.a. 480 16.8% 1.0% n.a. Example 36 35618.3% 0.4% 2.2% Example 37 356 6.7% 0.2% 0.1%In Vivo Animal Models

Known models of angiogenic tissue (rat matrigel model and rat Mat B IIImodel) were used to examine the ability of the peptide conjugatedultrasound conjugates to localize to and provide an image of angiogenictissue.

Animals: Female Fisher 344 rat (Charles River Laboratories, France)weighing 120 to 160 g were used for the MATBIII tumor implantation. MaleOFA rats (Charles River Laboratories, France) weighing 100 to 150 g wereused for Matrigel injection.

Anesthesia: Rats were anesthetized with an intramuscular injection (1mL/kg) of Ketaminol/xylazine (Veterinaria AG/Sigma) (50/10 mg/mL)mixture before implantation of Matrigel or MatBIII cells. For imagingexperiments, animals were anesthetized with the same mixture, plussubcutaneous injection of 50% urethane (1 g/kg).

Rat MATBIII tumor model: A rat mammary adenocarcinoma, designated 13762Mat B III, was obtained from ATCC(CRL-1666) and grown in McCoy's 5amedium+10% FCS. 1% glutamine and 1% pen/strep (Invitrogencat#15290-018). Cells in suspension were collected and washed in growthmedium, counted, centrifuged and resuspended in PBS or growth medium at1.10⁷ cells per mL. For tumor induction: 1×10⁶ cells in 0.1 mL wereinjected into the mammary fat pad of anesthetized female Fisher 344 rat.Tumors usually grow to a diameter of 5-8 mm within 8 days.

Rat matrigel model: Matrigel (400 μL) (ECM, Sigma, St Louis, Mo.)containing human bFGF (600 ng/mL) (Chemicon: ref: GF003) wassubcutaneously injected in the dorsal flank of each rat.

Matrigel solution was kept liquid at 4 C until injection. Immediatelyafter matrigel injection, the injection site was maintained closed for afew seconds with the hand in order to avoid leaking of the matrigel. Atthe body temperature, matrigel becomes gelatinous. Ten dayspost-injection, neoangiogenesis was observed in matrigel plug of rat andimaging experiment were performed.

In vivo ultrasound imaging: Mat B III tumor or matrigel imaging wasperformed using an ultrasound imaging system ATL HDI 5000 apparatusequipped with a L7-4 linear probe. B-mode pulse inversion at lowacoustic power (MI=0.05) was used to follow accumulation of peptideconjugated-microbubbles on the KDR receptor expressed on the endotheliumof neovessels. For the control experiments, an intravenous bolus ofunconjugated microbubbles or microbubbles conjugated to non-specificpeptide was injected. The linear probe was fixed on the skin directly online with the implanted tumors or matrigel plug and accumulation oftargeted bubbles was followed during thirty minutes.

In both models, a perfusion of SonoVue® was administrated beforeinjecting the test bubble suspension. This allows for the evaluation ofthe vascularization status; the video intensity obtained after SonoVue®injection is taken as an internal reference.

A baseline frame was recorded and then insonation was stopped during thebubble injection. At various time points after injection (1, 2, 5, 10,15, 20, 25, 30 minutes) insonation was reactivated and 2 frames of onesecond were recorded on a videotape.

Video frames from matrigel or Mat B III tumor imaging experiments werecaptured and analysed with the video-capture and Image-Pro Plus 2.0software respectively. The same rectangular Area of Interest (AOI)including the whole sectional area of the tumor or matrigel was selectedon images at different time points (1, 2, 5, 10, 15, 20, 25, 30minutes). At each time point, the sum of the video pixel inside the AOIwas calculated after the substraction of the AOI baseline. Results areexpressed as the percentage of the signal obtained with SonoVue, whichis taken as 100%. Similarly, a second AOI situated outside from matrigelor tumor, and representing the freely circulating contrast agent, isalso analysed.

Results

The results indicate that ultrasound contrast agents bearing KDR bindingmoieties of the invention localize to angiogenic (and thus KDRexpressing) tissue in animal models. Specifically, FIG. 81 shows uptakeand retention of bubble contrast in the tumor up to 30 minutes postinjection for suspensions of phospholipids stabilized microbubblesconjugated to KDR peptides of the invention prepared according toExample 35. In contrast, the same bubbles showed only transient (no morethan 10 minutes) visualization/bubble contrast in the AOI situatedoutside the tumor site. Similarly, FIG. 82 and FIG. 83 show uptake andretention of bubble contrast in the matrigel at up to 30 minutes postinjection for suspensions of phospholipids stabilized microbubblesconjugated to KDR peptides of the invention (e.g., SEQ ID NOS: 374 and294, respectively) prepared according to Example 35. In contrast, thesame bubbles showed only transient (no more than 10 minutes)visualization/bubble contrast in the AOI situated outside the matrigelsite.

Example 39 Enhancing the Serum Residence of KDR-Binding Peptides

Compounds that contain maleimide and other groups that can react withthiols react with thiols on serum proteins, especially serum albumin,when the compounds are injected. The adducts have serum life timessimilar to serum albumin, more than 14 days in humans for example.

Conjugation to Maleimide

Methods are available that allow for the direct synthesis ofmaleimide-labeled linear peptides encompassed by the present invention(Holmes, D. et al., 2000. Bioconjug. Chem., 11:439-444).

Peptides that include disulfides can be derivatized with maleimide inone of several ways. For example, a third cysteine can be added at thecarboxy terminus. The added cysteine is protected with protecting groupthat is orthogonal to the type of groups used for the cysteines that areto form the disulfide. The disulfide is formed by selectivelydeprotecting the intended cysteines and oxidizing the peptide. The finalcysteine is then deprotected and the peptide reacted with a large molarexcess of a bismaleimide. The resulting compound has one of themaleimides free to react with serum albumin or other thiol-containingserum proteins.

Alternatively, a cyclic peptide of the present invention is synthesizedwith a lysine-containing C-terminal extention, such as -GGGK (SEQ ID NO:262). Lysines of the KDR-binding motif are protected with ivDde and theC-terminal lysine is deprotected. This lysine is reacted with amaleimide-contining compound, such asN-[e-maleimidocaproyloxy]succinimide ester (Pierce Biotechnology,Rockford, Ill.) or N-(a-Maleimidoacetoxy)succinimide ester (PierceBiotechnology).

Conjugation to a Moiety that Binds Serum Albumin Non-Covelently

Polypeptides having a molecular weight less than 50-60 kDa are rapidlyexcreted. Many small molecules, such as fatty acids, bind to serumalbumin. Fatty acids containing 10 to 20 carbon atoms have substantialaffinity for serum albumin. Linear and branched fatty acids can be used.This binding in serum can reduce the rate of excretion. Using methodsknown in the art, serum-albumin-binding moieties can be conjugated toany one of the peptides herein disclosed. The serum-albumin-bindingmoiety can be joined to the KDR-binding peptide through a linker. Thelinker can be peptidic or otherwise, such as PEG. Linkers of zero toabout thirty atoms are preferred. It is preferred that the linker behydrophilic. The serum-albumin-binding moiety can be conjugated to theKDR-binding peptide at either end or though a side group of an appendedamino acid. Suitable side groups include lysine and cysteine. Suchcompounds can also comprise chelators for radionuclides, as discussedherein. A KDR-binding peptide joined to a serum-albumin-binding moietywill bind KDR.

Conjugation to PEG

Attachment of poly(ethyleneglycol) (PEG) to proteins and peptidesenhances the serum residence of these molecules. Attachment of PEG(linear or branched) to a KDR-binding peptide is expected givesubstantial enhancement of serum residence time. The molecular weight ofthe PEG should be at least 10 kDA, more preferably at least 20 kDa, andmost preferably 30 kDa or more. The PEG could be attached at the N- orC-terminus. Methods of attaching PEG to peptides are well known in theart (Roberts M. et al., 2002. Adv. Drug. Deliv. Rev., 54:459-476). PEGcan be attached to reactive side groups such as lysine or cysteine.

Fusion to Serum Protein

Proteins comprising serum albumin (SA) and other proteins have enhancedserum residence times. The amino-acid sequence of human SA (hSA) isshown in Table 22. Table 23 shows a fusion protein comprising:AGDWWVECRVGTGLCYRYDTGTGGGK(SEQ ID NO: 286)::PGGSGGEGGSGGEGGRPGGSEGGTGG::mature hSA:: GGSGGEGGSGGEGGSGPGEGGEGSGGRP::GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 294). The KDR-binding peptides areseparated from mature hSA by linkers that are rich in glycine to allowflexible spacing. One need not use all of hSA to obtain an injectableprotein that will have an enhanced serum residence time. Chemicalgroups, such as maleimide and alpha bromo carboxylates, react with theunpaired cysteine (residue 34) to form stable adducts. Thus, one canattach a single chelator to hSA fusion proteins so that the adduct willbind a radionuclide. One can prepare a chelator with a maleimide groupand couple that to hSA or an hSA derivative. Alternatively, hSA or anhSA derivative can be reacted with a bismaleimide and a chelatorcarrying a reactive thiol could be reacted with thebismaleimide-derivatized hSA.

Construction of genes that encode a given amino-acid sequence are knownin the art. Expression of HSA fusions in Saccharomyces cerevisiae isknown in the art (Sleep, D et al., 1991. Biotechnology (NY), 9:183-187).

Pretargeting Radioactivity or Toxins to KDR Expressing Tumors

Conventional radioimmune cancer therapy is plagued by two problems. Thegenerally attainable targeting ratio (ratio of administered doselocalizing to tumor versus administered dose circulating in blood orratio of administered dose localizing to tumor versus administered dosemigrating to bone marrow) is low. Also, the absolute dose of radiationor therapeutic agent delivered to the tumor is insufficient in manycases to elicit a significant tumor response. Improvement in targetingratio or absolute dose to tumor would be of great importance for cancertherapy.

The present invention provides methods of increasing active agentlocalization at a target cell site of a mammalian recipient. The methodsinclude, for example, a) administering to a recipient a fusion proteincomprising a targeting moiety and a member of a ligand-anti-ligandbinding pair; b) thereafter administering to the recipient a clearingagent capable of directing the clearance of circulating fusion proteinvia hepatocyte receptors of the recipient, wherein the clearing agentincorporates a member of the ligand-anti-ligand binding pair; and c)subsequently administering to the recipient an active agent comprising aligand/anti-ligand binding pair member.

Hexoses, particularly the hexoses galactose, glucose, mannose,mannose-6-phosphate, N-acetylglucosamine, pentamannosyl phosphate,N-acetylgalactosamine, thioglycosides of galactose, and mixtures thereofare effective in causing hepatic clearance. Binding of sugars to hepaticreceptors is not, however, the only means of directing a molecule to theliver.)

Clearance of carcinoembryonic antigen (CEA) from the circulation is bybinding to Kupffer cells in the liver. We have shown that CEA binding toKupffer cells occurs via a peptide sequence YPELPK representing aminoacids 107-112 of the CEA sequence. This peptide sequence is located inthe region between the N-terminal and the first immunoglobulin like loopdomain. Using native CEA and peptides containing this sequence complexedwith a heterobifunctional crosslinking agent and ligand blotting withbiotinylated CEA and NCA we have shown binding to an 80 kD protein onthe Kupffer cell surface. This binding protein may be important in thedevelopment of hepatic metastases. (Thomas, P. et al., 1992. Biochem.Biophys. Res. Commun., 188:671-677

To use YPELPK (SEQ ID NO: 498) as a clearance agent, one fuses thissequence via a linker to a moiety that binds the fusion protein (Ab).For example, if the Ab has affinity for DOTA/Re, one would make aderivative having YPELPK attached to DOTA/Re; for example,rvYPELPKpsGGG-DOTA. ‘rvYPELPKps’ is a fragment of CEA that includes theYPELPK sequence identified by Thomas et al. Any convenient point on DOTAcan be use for attachment. RVYPELPKPSGGG-DOTA/cold Re (SEQ ID NO: 499)would then be used as a clearing agent. The Fab corresponding to thefusion Ab would have affinity for the clearing agent of Kd<100 nM,preferably Kd<10 nM, and most preferably Kd<1 nM.

The therapeutic agent would contain DOTA/¹⁸⁵Re. In a preferredembodiment, the therapeutic agent would contain two or more DOTAmoieties so that the Ab immobilized on the tumor would bind the bis-DOTAcompound with high avidity. The two DOTA moieties would preferably beconnected with a hydrophilic linker of ten to thirty units of PEG. PEGis a preferred linker because it is not degraded, promotes solubility.Ten to thirty units of PEG is not sufficient to give the bis DOTAcompound a very long serum residence time. A half-life of 30 minutes to10 hours is acceptable. The serum half life should be longer than theradioactive half life of the radionuclide used so that most of theradiation is delivered to the tumor or to the external environment.

In one embodiment, a “fusion protein” of the present invention comprisesat least one KDR-binding peptide fused to the amino terminus or thecarboxy terminus of either the light chain (LC) or the heavy chain (HC)of a human antibody. Optionally and preferably, two or more KDR-bindingpeptides are fused to the antibody. The antibody is picked to have highaffinity for a small molecule that can be made radioactive or have atoxin attached. Preferably, the affinity of the Fab corresponding to theAb has affinity for the small molecule with Kd less than 100 nM, morepreferably less than 10 nM, and most preferably less than 1 nM. Thesmall molecule could be a chelator capable of binding a usefulradioactive atom, many of which are listed herein. The small moleculecould be a peptide having one or more tyrosines to which radioactiveiodine can be attached without greatly affecting the binding property ofthe peptide.

Any KDR-binding peptide (KDR-BP) of the present invention can be fusedto either end of either chain of an antibody that is capable of bindinga small radioactive compound.

Useful embodiments include:

1) KDR-BP#1::link::LC/HC,

2) LC::link::KDR-BP#1/HC,

3) LC/KDR-BP#1::link::HC,

4) LC/HC::link::KDR-BP#1,

5) KDR-BP#1::link1::LC::link2::KDR-BP#2/HC,

6) LC/KDR-BP#1::link1::HC::link2::KDR-BP#2,

7) KDR-BP#1::link1::LC/KDR-BP#2::link2::HC,

8) KDR-BP#1::link1::LC/HC::link2::KDR-BP#2,

9) LC::link1::KDR-BP#1/KDR-BP#2::link2::HC,

10) LC::link1::KDR-BP#1/HC::link2::KDR-BP#2,

11) KDR-BP#1::link1::LC::link2::KDR-BP#2/KDR-BP#3::link3::HC,

12) KDR-BP#1::link1::LC::link2::KDR-BP#2/HC::link3::KDR-BP#3,

13) KDR-BP#3::link3::LC/KDR-BP#1::link1::HC::link2::KDR-BP#2,

14) LC::link3::KDR-BP#3/KDR-BP#1::link1::HC::link2::KDR-BP#2, and

15)KDR-BP#1::link1::LC::link2::KDR-BP#2/KDR-BP#3::link3::HC::link4::KDR-BP#4.

In cases (5)-(15), the linkers (shown as “link1”, “link2”, “link3”, and“link4”) can be the same or different or be absent. These linkers, ifpresent, are preferably hydrophilic, protease resistant, non-toxic,non-immunogenic, and flexible. Preferably, the linkers do not containglycosylation sites or sequences known to cause hepatic clearance. Alength of zero to fifteen amino acids is preferred. The KDR-bindingpeptides (KDR-BP#1, #2, #3, and #4) could be the same or different. Ifthe encoded amino-acid sequences are the same, it is preferred that theDNA encoding these sequences is different.

Since antibodies are dimeric, each fusion protein will present twocopies of each of the fused peptides. In case (15), there will be eightKDR-BPs present and binding to KDR-displaying cells should be highlyavid. It is possible that tumor penetration will be aided by moderateKDR affinity in each of the KDR-BPs rather than maximal affinity.

One group of preferred embodiments have SEQ ID NO: 294 as one of theKDR-BPs and SEQ ID NO: 286 as the other. For example, in case (7)(KDR-BP#1::link1::LC/KDR-BP#2::link2::HC), KDR-BP#1 is SEQ ID NO: 294and KDR-BP#2 is SEQ ID NO: 286 and link1 is between 10 and 20 aminoacids and link2 is also between ten and twenty amino acids. A suitablesequence for link1 is GGSGGEGRPGEGGSG (SEQ ID NO: 491) and a suitablesequence for link2 is GSESGGRPEGGSGEGG (SEQ ID NO: 492). Other sequencesrich in Gly, Ser, Glu, Asp, Thr, Gln, Arg, and Lys are suitable. Toreduce the risk of proteolysis, it is preferred to follow Arg or Lyswith Pro. To avoid difficulties in production and poor solubility, it ispreferred to avoid long stretches (more than twelve) of unchargedresidues. Since the peptides are displayed at the amino termini of LCand HC, the combined linker length will allow them to bind to KDRsimultaneously. Additionally, in case(15)(KDR-BP#1::link1::LC::link2::KDR-BP#2/KDR-BP#3::link3::HC::link4::KDR-BP#4),KDR-BP#1 and KDR-BP#2 are SEQ ID NO: 294 and KDR-BP#3 and KDR-BP#4 areSEQ ID NO: 29. Link1 and link3 are 10 to 20 amino acids and link2 andlink4 are each 15 to 30 amino acids. Link2 and link4 are longer becausethey need to allow a peptide on the carboxy terminus of LC to reach apeptide on the carboxy terminus of HC.

The fusion protein is produced in eukaryotic cells so that the constantparts of the HC will be glycosylated. Preferably, the cells aremammalian cells, such as CHO cells.

The fusion proteins are injected into a patient, and time is allowed forthe fusion protein to accumulate at the tumor. A clearing agent isinjected so that fusion protein that has not become immobilized at thetumor will be cleared. In previous pretargeting methods, the antibodycombining site has been used to target to the tumor and biotin/avidin orbiotin/streptavidin has been used to attach the radioactive or toxicagent to the immobilized antibody. The biotin/avidin or streptavidinbinding is essentially irreversible. Here we fuse a target-bindingpeptide to the antibody that is picked to bind a radioactive or toxicagent. Because the fusion protein contains 2, 4, 6, or 8 KDR-BPs,binding of the fusion protein to the tumor is very avid. A clearingagent that will cause fusion protein not immobilized at the tumor toclear can be administered between 2 and 48 hours of the injection of thefusion protein. Because the clearance agent is monomeric in the moietythat binds the antibody, complexes of clearance agent and immobilizedfusion protein will not have very long life times. Within 4 to 48 hoursof injecting clearance agent, the immobilized antibody will have lostany clearance agent that binds there. The active agent is, preferably,dimeric in the moiety that binds the fusion protein. The active agent isinjected between 2 and ˜48 hours of injection of clearance agent.

TABLE 22 Amino-acid sequence of Mature HSA from GenBank entry AAN17825DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHVKLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATLRETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEVDVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKRYKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKCASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTKVHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCEKPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYAEAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYKTTLEKCCAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGEYKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKHPEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTESLVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKERQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCKADDKETCFAE EGKKLVAASR AALGL (SEQ ID NO: 500)

TABLE 23 SEQ ID NO: 286::linker1::HSA::linker2::SEQ ID NO: 294AGDWWVECRVGTGLCYRYDTGTGGGK PGGSGGEGGSGGEGGRPGGSEGGTGGDAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHVKLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATLRETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEVDVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKRYKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKCASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTKVHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCEKPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYAEAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYKTTLEKCCAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGEYKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKHPEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTESLVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKERQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASR AALGLGGSGGEGGSGGEGGSGPGEGGEGSGGRP GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 501)

Example 40 Synthesis of Dimers D30 and D31

Preparation ofAc-VCWEDSWGGEVCFRYDPGGGK{[PnAO6-Glut-K(-Glut-JJ-NH(CH₂)₄—(S)—CH(Ac-AQDWYYDEILJGRGGRGGRGG-NH)C(═O)NH₂]—NH₂}—NH₂:Dimer D30

Preparation of Ac-VCWEDSWGGEVCFRYDPGGGK[PnAO6-Glut-K]—NH₂ (Compound 3;FIG. 87A)

Ac-VCWEDSWGGEVCFRYDPGGGK[K(iV-Dde)]-NH₂ [(1), comprising SEQ ID NO: 494,is a SEQ ID NO: 374 derivative; specifically Acetyl-(SEQ ID NO: 374,5-21)-GGGK[K(iV-Dde), 48 mg] was prepared by the procedures of Method 5.The compound was dissolved in DMF (0.85 mL) and treated with compound Band DIEA (7 μL) was added to maintain the basicity of the reactionmixture. The progress of the reaction was monitored by HPLC and massspectroscopy. At the completion of the reaction (20 h), the volatileswere removed in vacuo. The residue, which consists of a compound 2 (SEQID NO: 374, 5-21) derivative, specifically Acetyl-(SEQ ID NO: 374,5-21)-GGGK[(PnAO6-Glut-)K(iV-Dde)]-NH₂), was treated with 10% hydrazinein DMF (5 μL) for 10 min. HPLC analysis and mass spectroscopy indicatedthe completion of the reaction. The mixture then was applied directly toa Waters Associates XTerra MSC18 preparative HPLC column (50 mm×19 mmi.d.) and purified by elution with a linear gradient of acetonitrileinto water (both containing 0.1% TFA) to provide 11 mg of pure Compound3.

Preparation of the Dimer D30 from Compound 3 andAc-AQDWYYDEIL-Adoa-GRGGRGGRGGGK(Adoa-Adoa)-NH₂ (Compound 4 (comprisingSEQ ID NO: 617 with Modified Lysine Side Chains; Based on the PeptideBinding Moiety of SEQ ID NO: 376))

Disuccinimidyl glutarate (12 mg) was dissolved in DMF (500 μL), and DIEAwas added (1 μL). Compound 3 in DMF was added into the DMF solution ofdisuccinimidyl glutarate/DIEA. The mixture was stirred for 2.5 h. HPLCand mass spectroscopy indicated the completion of the reaction. Thevolatiles were removed in vacuo and the residue was washed with ether(3×) to remove the unreacted bis-NHS ester. The residue was dried,re-dissolved in anhydrous DMF and treated with the Compound 4,Ac-AQDWYYDEIL-Adoa-GRGGRGGRGGGK(Adoa-Adoa)-NH₂, which was prepared byMethod 5 and Method 8, in the presence of 2 equivalents of DIEA. Thereaction was allowed to proceed for 20 h. The mixture then was applieddirectly to a Waters Associates MSC18 reverse phase preparative (50mm×19 mm i.d.) HPLC column and purified by elution with a lineargradient of acetonitrile into water (both containing 0.1% TFA) toprovide 2 mg of D30 (For purification and structure of D30, see belowand also FIGS. 87B and C, respectively).

Synthesis ofAc-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK[SGS-Glut-SGS-(S)—NH(CH₂)₄—CH(Biotin-JJ-NH)—C(═O)]—NH₂]—NH₂:D31

Preparation of Monomer Compound 2 and Monomer Compound 4

See FIG. 88B.

Synthesis of Monomer Peptide 1 and Monomer Peptide 3

Monomer Peptide 1 comprises SEQ ID NO: 378 with the followingmodification: it is an Ne22-iV-Dde-SEQ ID NO: 378 peptide.

Monomer peptide 3 comprises SEQ ID NO: 370, and is a derivative of SEQID NO: 337. It is an Ne25-iV-Dde-SEQ ID NO: 370 peptide.

Synthesis of the monomers 1 and 3 were carried out using the proceduresof Method 5 for the ABI 433A synthesizer.

Synthesis of Monomer Peptide 2 and Monomer Peptide 4

See FIGS. 88A and 88B.

Appendage of Biotin-JJ, Lys, Gly and Ser onto Compounds 1 and 3 was doneby SPPS manually using the appropriate Fmoc amino acids, Biotin-JJ andFmoc-J (J=8-amino-3,6-dioxaoctanoic acid) according to the procedures ofMethods 6, 7, 8, 9 and 10. Cleavage of the peptides from the resin,processing of the crude peptides was carried out as described in Method1 for the synthesis of peptides. Cyclization of the cysteine moieties toform the cyclic disulfide peptides was performed by the procedures ofMethod 9.

Purification of the peptides was carried out using a Shimadzu LC-10AHPLC system and a YMC C-18 ODS preparative HPLC column employing alinear gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueousTFA. Pure fractions were combined and lyophilized.

The dimer D31 was prepared using monomer compound 4 to generate, insitu, the activated monomer compound 5, which was then reacted withmonomer compound 2 using the procedures described in Method 13,entitled: ‘Preparation of Heterodimer Containing Constructs’. The crudecompound D31 was purified by preparative reverse phase HPLC using aWaters-YMC C-18 ODS column to provide 10 mg of the dimer D31.

Example 41 In Vitro Competition Experiments on KDR-Transfected Cells

The following experiment assessed the specificity of the binding ofpeptide-conjugated microbubbles to KDR-expressing cells.

Protocol:

293H cells were transfected with KDR cDNA. The transfected cells wereincubated with a suspension of peptide-conjugated microbubbles inpresence or absence of the corresponding free peptide (at 100, 30, 10,3, 1, 0.3, 0.1 μM). Microbubbles were conjugated to a SATA-modifiedpeptide comprising SEQ ID NO: 480, a SATA-modified peptide comprisingSEQ ID NO: 356, or a SATA-modified peptide comprising SEQ ID NO: 356 anda JJ linker. Competition was also performed using the correspondingnon-binding or control free peptide as competing compound. At the end ofthe incubation, the transfected cells were rinsed three times in PBS andexamined under a microscope. Binding of the conjugated bubbles wasquantified and expressed as percent of surface covered by the targetedmicrobubbles.

Results:

All the KDR-conjugated microbubbles were competed off by thecorresponding free KDR-specific peptide whereas the presence of controlpeptide had no effect. Example of curves obtained by plotting thefraction of residual binding as a function of the competitorconcentration are shown in FIG. 89.

Example 42 In Vitro Competition Experiments on KDR-Transfected Cells

The following experiment assessed the specificity of the binding ofpeptide-conjugated microbubbles to KDR-expressing cells.

Protocol:

293H cells were transfected with KDR cDNA. The transfected cells wereincubated with a suspension of peptide-conjugated microbubbles inpresence or absence of the corresponding free peptide (between 100 μM to3 nM). Competition was also performed using a non-binding peptide ascompeting compound. At the end of the incubation, the transfected cellswere rinsed three times in PBS and examined under a microscope. Bindingof the conjugated bubbles was quantified and expressed as percent ofsurface covered by the targeted microbubbles.

Results:

Microbubbles conjugated to KDR-specific dimer (D23) or monomer (SEQ IDNO: 338) molecules were competed off by the corresponding freeKDR-specific peptide whereas the presence of control peptide had noeffect. Example of curves obtained by plotting the fraction of residualbinding as a function of the competitor concentration are shown in FIG.90.

In Vitro Competition Experiments on KDR-Transfected Cells

The following experiment compares the binding efficiency of monomers anddimers conjugated to microbubbles on KDR-transfected cells.

Protocol:

293H cells were transfected with KDR cDNA. The transfected cells wereincubated with a suspension of microbubbles conjugated to differentpeptides (monomers or dimers) in presence or absence of increasingconcentrations of free dimer (at 1000, 300, 100, 30, 10, 3, 1 nM). Atthe end of the incubation, the transfected cells were rinsed three timesin PBS and examined under a microscope. Binding of the conjugatedbubbles was quantified and expressed as percent of surface covered bythe targeted microbubbles.

Results:

Microbubbles conjugated to D23 were more resistant to competition andless easily displaced by the corresponding free dimeric peptide thanKDR-specific monomer-conjugated microbubbles conjugated to SEQ ID NO:338 or SEQ ID NO: 376. Representative curves obtained by plotting thefraction of residual binding as a function of the competitorconcentration are shown in FIG. 91.

Example 43 In Vitro Binding of Heteromultimers and Dimers Compared toMultimeric Monomers

The following experiment aims at comparing the binding efficiency ofmixed monomers, dimers and monomers conjugated to microbubbles in theKDR-transfected cells assay.

Protocol:

Microbubbles were conjugated to either a dimer (D23) or two differentpeptides monomers (SEQ ID NO: 294 or SEQ ID NO: 480). A fourthconjugation reaction was performed using equal quantities of eachmonomer (and the same total peptide load). 293H cells were transfectedwith KDR cDNA. The transfected cells were incubated with the same numberof targeted microbubble and in presence of 50% human serum. At the endof the incubation, the transfected cells were rinsed three times in PBSand examined under a microscope. Binding of the conjugated bubbles wasquantified and expressed as percent of surface covered by the targetedmicrobubbles.

Results:

As shown in FIG. 92, microbubbles conjugated with SEQ ID NO: 294 boundpoorly compared with microbubbles conjugated with SEQ ID NO: 480 ordimer D23. Surprisingly, microbubbles conjugated to D23 boundequivalently to those conjugated to SEQ ID NO: 480 although D23 has halfthe load. Moreover, the “mixed monomer” conjugated microbubbles, whichalso have half the SEQ ID NO: 480 load, bound as well as microbubblesconjugated with SEQ ID NO: 480 or D23. These results show the increasedbinding capacity of heteromultimers.

Example 44 Blocking VEGF-Enhanced Peritoneal Vascular Permeability witha Heterodimeric Peptide

In this example, the ability of heterodimer D10 to inhibit the enhancedvascular permeability caused by VEGF injected into the peritoneum ofnude mice is demonstrated.

Protocol

Male balb/c nu/nu mice were injected intraperitoneally with 2 mL vehicle(1% bovine serum albumin in 95% saline/5% DMSO), vehicle+1.2 nM VEGF₁₆₅,or vehicle+1.2 nM VEGF₁₆₅+20 μM D10. Immediately after, the mice wereinjected with Evan's Blue Dye (0.5% in saline, 4 mL/kg) i.v. via theirtail veins. After 60 min, mice were sacrificed by CO₂ asphyxiation andthe peritoneal fluid was retrieved. After centrifuging the samplesbriefly, the absorbance at 590 nm was measured for each.

Results

As shown in FIG. 93, VEGF, when added to the fluid injectedintraperitoneally, significantly increased the dye leakage into theperitoneum, and this increase was substantially blocked by including D10with the VEGF.

Example 45 Mouse Xenograft Tumor Model of Human Colon Cancer

This example assesses the effects of dimer D6 that has been processedinto biodegradable sustained release pellets. Since D6 has a half-lifeon the order of 1 hour, a way of improving the residence time in serawas sought. The compound is formulated into a sustained release formatso that greater therapeutic benefit to animal models is observed.

The effect of D6 on the tumor model is determined, for example, bymeasuring tumor size with and without treatment. Additionally, theeffect of D6, engineered to have a longer residence time in sera, iscompared to the effect of unmodified D6 (see Example 39).

Briefly, 140 nude mice are injected subcutaneously with the cell line,SW-480. Tumors are measured, and when tumors reach 100-200 mg, 100animals are selected and randomized into 10 study groups of 10 animalseach. The overall study is summarized in Table 24 below. The dosingschedule follows the chart shown in Table 25. Tumor measurements aretaken on each animal twice a week during the normal workweek.Measurements are made by hand-held vernier caliper. Body weights andtumor measurements are recorded twice a week. This study is based on atypical four week study from beginning of dosing and includes removal of30 tumors.

TABLE 24 D6 Mouse Tumor Study Cell line SW-480, human colon carcinoma 5× 10⁶, subcutaneous Test Animal nude mouse (CRL: NU/NU = nuBR) female n= 10/test group Study Initiation >6 weeks age Tumor ~100 −/+ 50 mgControl 1. untreated 2. Vehicle 3. Placebo pellet 4. Cisplatin TestArticle D6 0.5 mg/kg/d × 21 d 2.0 mg/kg/d × 21 d 2.0 mg/kg/d × 21 dpellet Test Article Form 1. solution for injection (PBS, IP) 2.sustained release pellet (nominal 21 day, subcutaneous) Primaryendpoints 1. tumor growth 2. histopathology (necropsy) Supplementarymeasures 1. angiogenesis (CD-31+) (representative samples) 2. cellproliferation (PCNA) 3. circulating D6 4.

TABLE 25 Treatment Vehicle D6 Dose Cisplatin Dose Group n AdministrationAdministration Administration 1 10 — — — 2 10 PBS — — 1 IP inj/d, 21 d 310 — 0.5 mg/kg/day — 1 IP inj/d, 21 d 4 10 — 2.0 mg/kg/day — 1 IP inj/d,21 d 5 10 Vehicle pellet — — (1), sc 6 10 — 2.0 mg/kg/day — pellet (1),sc 7 10 — — [6 mg/kg] 1 IV inj/2 days, to 5 Ttl 8 10 — 2.0 mg/kg/day [6mg/kg] pellet (1), sc 1 IV inj/2 days to 5 Ttl 9 10 — 2.0 mg/kg/day [3mg/kg] pellet (1), sc 1 IV/2 days, to 5 Ttl 10 10 — 2.0 mg/kg/day [1mg/kg] pellet (1), sc 1 IV/2 days, to 5 Ttl

Example 46

The following example describes the preparation of an ultrasoundcontrast agent conjugated to a KDR-binding heterodimer of the inventionand the ability of the heterdimer conjugated contrast agent to localizeto KDR-expressing cells in vitro and angiogenic tissue in vivo.

Preparation of Derivatized Microbubbles for Peptide Conjugation

200 mg of DSPC (distearoylphosphatidylcholine), 275 mg of DPPG.Na(distearoylphosphatidylglycerol sodium salt) and 25 mg of N-MPB-PE weresolubilized at 60° C. in 50 mL of Hexan/isopropanol (42/8). The solventwas evaporated under vacuum, and then PEG-4000 (35.046 g) was added tothe lipids and the mixture was solubilized in 106.92 g of t-butylalcohol at 60° C., in a water bath. The solution was filled in vialswith 1.5 mL of solution. The samples were rapidly frozen at −45° C. andlyophilized. The air in the headspace was replaced with a mixture ofC₄F₁₀/Air (50/50) and vials capped and crimped. The lyophilized sampleswere reconstituted with 10 mL saline solution (0.9% —NaCl) per vial,yielding a suspension of phospholipids stabilized microbubbles.

Peptide Conjugation

D23 was conjugated with a preparation of microbubbles as abovedescribed, according to the following methodology. The thioacetylatedpeptide (200 μg) was dissolved in 204, DMSO and then diluted in 1 ml ofPhosphate Buffer Saline (PBS). This solution was mixed to theN-MPB-functionalized microbubbles dispersed in 18 mL of PBS-EDTA 10 mM,pH 7.5, and 2 mL of deacetylation solution (50 mM sodium phosphate, 25mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) was added. The headspace wasfilled with C₄F₁₀/Air (50/50) and the mixture was incubated for 2.5hours at room temperature under gentle agitation (rotating wheel), inthe dark. Conjugated bubbles were washed by centrifugation. Similarly,the monomer peptides making up D23 were separately conjugated to twodifferent microbubble preparations according to the methodologydescribed above.

In Vitro Assay on Transfected Cells

The ability of phospholipid stabilized microbubbles conjugated topeptides and heteromultimeric peptide constructs of the invention tobind to KDR-expressing cells was assessed using 293H cells transfectedto express KDR.

Transfection of 293H cells on Thermanox® Coverslips

293H cells were transfected with KDR DNA as set forth in Example 5. Thetransfected cells were incubated with a suspension of peptide-conjugatedmicrobubbles prepared as described above. For the incubation with thetransfected cells a small plastic cap is filled with a suspensioncontaining 1 to 3×10⁸ peptide-conjugated microbubbles and the capcovered with an inverted Thermanox® coverslip is placed so that thetransfected cells are in contact with the conjugated microbubbles. Afterabout 20 min at room temperature, the coverslip is lifted with tweezers,rinsed three times in PBS and examined under a microscope to assessbinding of the conjugated microbubbles.

Determination of the Percent of Surface Covered by Microvesicles

Images were acquired with a digital camera DC300F (Leica) and thepercent of surface covered by bound microbubbles in the imaged area wasdetermined using the software QWin (Leica Microsystem AG, Basel,Switzerland). Table 26 shows the results of the binding affinity(expressed as coverage % of the imaged surface) of targetedmicrovesicles of the invention to KDR transfected cells, as compared tothe binding of the same targeted microvesicles to Mock-transfectedcells.

TABLE 26 Conjugated microbubbles % of covered prepared as describedabove surface Peptide code Batch Id KDR Mock SEQ ID NO: 294 BG1979T023.5% 0.9% Derivative SEQ ID NO: 480 BG1980T02 16.8% 1.0% Derivative D23(dimer) BG2002T02 22.9% 3.3% SEQ ID NO. 294/ BG1958T02 12.9% 0.8% SEQ IDNO: 480 Deriv.

Where the SEQ ID NO: 294-derived sequence and the SEQ ID NO: 480-derivedsequence are separately attached to phospholipid stabilized microbubblesas monomers the resulting preparations achieve binding of the bubbles toKDR transfected cells in vitro to a different extent (3.5% and 16.8%).When a preparation of phospholipid stabilized microbubbles resultingfrom the addition of equal quantities of each of these peptide monomers(but the same total peptide load) is tested in the same system, 12.9%binding is achieved. Binding is a little more than the average of thetwo but as it is achieved with two sequences that bind to differentsites on the target will be more resistant to competition at one orother of the sites on the target. However, for D23, the dimer, bindingis increased to 22.9% (with the same peptide load). These resultsindicate that hetromultimers of the invention permit increased bindingand increased resistance to competition.

In Vivo Animal Models

A known model of angiogenic tissue (the rat Mat B III model) was used toexamine the ability of phospholipids-stabilized microbubbles conjugatedto a heteromultimer of the invention to localize to and provide imagesof angiogenic tissue.

Female Fisher 344 rat (Charles River Laboratories, France) weighing 120to 160 g were used for the MATBIII tumor implantation. Male OFA rats(Charles River Laboratories, France) weighing 100 to 150 g were used forMatrigel injection.

Anesthesia

Rats were anesthetized with an intramuscular injection (1 mL/kg) ofKetaminol®/xylazine (Veterinaria AG/Sigma) (50/10 mg/mL) mixture beforeimplantation of Matrigel or MatBIII cells. For imaging experiments,animals were anesthetized with the same mixture, plus subcutaneousinjection of 50% urethane (1 g/kg).

Rat MATBIII Tumor Model

A rat mammary adenocarcinoma, designated 13762 Mat B III, was obtainedfrom ATCC(CRL-1666) and grown in McCoy's 5a medium+10% FCS. 1% glutamineand 1% pen/strep (InVitrogen cat#15290-018). Cells in suspension werecollected and washed in growth medium, counted, centrifuged andresuspended in PBS or growth medium at 1×10⁷ cells per mL. For tumorinduction: 1×10⁶ cells in 0.1 mL were injected into the mammary fat padof anesthetized female Fisher 344 rat. Tumors usually grow to a diameterof 5-8 mm within 8 days.

In Vivo Ultrasound Imaging

Tumor imaging was performed using an ultrasound imaging system ATL HDI5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversionat low acoustic power (MI=0.05) was used to follow accumulation ofpeptide conjugated-microbubbles on the KDR receptor expressed on theendothelium of neovessels. For the control experiments, an intravenousbolus of unconjugated microbubbles or microbubbles conjugated tonon-specific peptide was injected. The linear probe was fixed on theskin directly on line with the implanted tumors and accumulation oftargeted bubbles was followed during thirty minutes.

A perfusion of SonoVue® was administrated before injecting the testbubble suspension. This allows for the evaluation of the vascularizationstatus and the video intensity obtained after SonoVue® injection istaken as an internal reference.

A baseline frame was recorded and then insonation was stopped during theinjection of the microbubbles. At various time points after injection(1, 2, 5, 10, 15, 20, 25, 30 minutes) insonation was reactivated and 2frames of one second were recorded on a videotape.

Video frames from tumor imaging experiments were captured and analysedwith the video-capture and Image-Pro Plus 2.0 software respectively. Thesame rectangular Area of Interest (AOI) including the whole sectionalarea of the tumor was selected on images at different time points (1, 2,5, 10, 15, 20, 25, 30 minutes). At each time point, the sum of the videopixel inside the AOI was calculated after the subtraction of the AOIbaseline. Results are expressed as the percentage of the signal obtainedwith SonoVue®, which is taken as 100%. Similarly, a second AOI situatedoutside the tumor, and representing the freely circulating contrastagent, is also analyzed.

FIG. 94 shows uptake and retention of bubble contrast in the tumor up to30 minutes post injection for suspensions of phospholipid stabilizedmicrobubbles conjugated to a heteromultimeric construct of the inventionprepared as described above (D23). In contrast, the same bubbles showedonly transient (no more than 10 minutes) visualization/bubble contrastin the AOI situated outside the tumor site.

Example 47

The study described below was performed to assess the ability ofcontrast agents containing representative KDR-binding peptides describedherein to detect, monitor and assess the anti-angiogenic therapeuticeffect of therapeutic agents such as Sunitinib (a potent tyrosine kinaseinhibitor, which inhibits KDR).

Material and Methods

Four (4) female OFA rats (Charles River, Lyon, France) wereinvestigated. At the specific age of 42 days, animals receivedintraperitoneal injection of N-methyl-N-nitrosourea 50 mg/kg (NMU,Sigma-Aldrich, ref D3254) prepared in 0.9% NaCl, in order to develop aKDR-expressing tumor in the animals (see latency period infra) Weight ofthe animals was 241.8±6.3 g when entering the protocol. Animals receivedfood and water (tap water) ad libitum.

Study Treatment

Sunitinib malate salt (LC Laboratories, ref S-8803) was dissolved inNaCl 0.9% containing 10% polyethylene glycol 300 (PEG 300,Sigma-Aldrich, ref 90878), 0.5% polysorbate 80 (Tween 80, Sigma-Aldrich,ref) pH 3.75. Stock solution was made weekly and stored at 4° C. Ratsreceived daily oral gavage of 53.6 mg/kg of sunitinib malate salt, whichcorresponds to 40 mg/kg of sunitinib.

Animal Preparation

Gaseous anesthesia of the rat was achieved by Isoflurane inhalation 5%in 1 L/min oxygen enriched (95%) air. At the level of the tumor site,skin was gently shaved and an ultrasound coupling gel (Aquasonic 100,Parker, USA) was applied between the clinical ultrasound probe and theskin.

Imaging Examination

Ultrasound imaging was performed using a clinical ultrasound device(Philips iU22, vision 2008, Philips Medical System, Eindhoven, TheNetherlands), operating in a contrast-specific imaging mode. The linearprobe L12-5 was used.

Experimental Procedure

Dosing of Animals

Vials of a contrast agent containing D5 were reconstituted in 2 mlglucose 5%. D5 is described in more detail in Examples 50 and 51. Asdescribed in Example 51, D5 includes the peptide monomers (12) (SEQ IDNO. 277) and (13) (SEQ ID NO. 337) which form dimer (16) (D5).Lipopeptides comprising the dimer D5 were prepared as described inExample 52. The lyophilized precursor of the contrast agent was obtainedaccording to the procedure illustrated in Example 59a, by replacingDSPE-PEG2000 with DPPE-PEG5000 (2.3 mg) and admixing it with 3.3. mg ofthe dimeric phospholipid conjugate (11) of FIG. 5 in 60 mL of adistilled water, containing 10% PEG4000. Stearate was further replacedby palmitic acid (1.0 mg) and admixed with DSPC (59 mg) in cyclooctane(4.8 ml). Emulsification was effected at 12′000 rpm (five minutes). Thelyophilizate was reconsistituted as described above. The contrast agentwas quickly injected through the catheter placed in the caudal vein atthe dose of 2 μl gas per kg. The injection volume ranged from 49 to 61μl followed by a flush of 100 μl saline. Injections were performed at aconstant rate of 4 ml/min.

Echographic Evaluations

Tumor size was assessed in fundamental-B mode using calipers.

For each injection, tumor imaging was performed in contrast modestarting 10 sec before contrast injection until 10 to 14 minutes afterinjection

Quantification of Tumor Contrast Enhancement and Targeting

Contrast enhancement was quantified from Dicom files using an in-housequantification and providing, among other parameters, mean echo-signalvalues in areas of interest as a function of time. The amplitude of theecho signal was expressed as linearized echo amplitude values.Individual frames were analyzed, time-intensity curves were generatedand Maximum intensity (Imax) was determined as an index of tumorvascularization and late phase opacification (LPO) was determined at 10min after injection in the median plane of the tumor.

Results

Latency for tumor appearance ranged between 44 days and 93 days afterNMU-treatment for the four tested rats. When the lesion was detected,tumor size, assessed by fundamental-B mode was around 0.3 cm² (largestcross section). Performing in vivo imaging with D5-containingmicrobubbles showed that Imax, an index of tumor vascularization, wasquite variable throughout the follow up of tumor growth whereas LPO,which correlates with the expression of KDR receptor, was more stableover the duration of the protocol.

In rats no. 1, no. 2 and no. 3, treatment with sunitinib (a potenttyrosine kinase inhibitor, which inhibits KDR) was initiated on day 106after NMU treatment, at a dose of 40 mg/kg/day. Tumor size wasdetermined in fundamental B-mode. Values were multiplied by 10 to fitwith Y abscissa scale. Imax and LPO were measured using contrast modeand after injection of the D5-containing contrast agent.

The results for rat no. 1 are shown in FIG. 98 and in Table 28 below.

TABLE 28 day of imaging Imax LPO Area *10 79 436.0 12 5.60 85 1294.0 2013.90 91 1220.0 24 18.10 105 1090.0 20 21.80 108 966.0 14 20.80 1151095.0 10 11.00 116 931.5 8 10.10

Sunitinib treatment period is indicated by the red box in FIG. 98.Treatment started on day 106.

The results for rat no. 2 are shown in FIG. 99 and in Table 29 below.

TABLE 29 day of imaging Imax LPO Area *10 79 930 25 3.40 85 1629 31 7.2093 2660 54 16.80 105 1523 36 26.90 108 1882 10 26.70 109 406 5 20.20

Sunitinib treatment period is indicated by the red box in FIG. 99.Treatment started on day 106. On day 109, tumor was harvested andprocessed for immunohistochemistry. FIG. 103 providesimmunohistochemistry images for this sample.

The results for rat no. 3 are shown in FIG. 100 and in Table 30 below.

TABLE 30 day of imaging Imax LPO Area *10 93 2600 30 9.1 101 3456 24 9.8105 2900 30 10.8 108 3200 10 7.5 114 1327 7 2.9 116 1524 5 3.0

Sunitinib treatment period is indicated by the red box in FIG. 100.Treatment started on day 106. The results for rat no. 4 are shown inFIG. 101 and in Table 31 below.

TABLE 31 day of imaging Imax LPO Area *10 44 127 5 3.370 50 441 2 4.57056 439 9 5.000 63 912 18 8.590 78 825 20 14.500

On day 78, tumor was harvested and processed for immunohistochemistrystaining FIG. 104 provides immunohistochemistry images for this sample.

48 hours after treatment (day 108) tumor size remained unchanged in ratsno. 1 and no. 4 (respectively −4.6% and −0.7% when expressed as apercentage of the tumor prior sunitinib treatment). In rat no. 3 therewas a reduction of tumor size by 30%. Imax was slightly decreased in ratno. 1 (−11%) and increased in both rats no. 2 and no. 3 (+23% and +11%respectively). Finally, the measurement of LPO, i.e. targetedmicrobubbles bound to KDR receptor, revealed a strong decrease in all 3rats 24 hours after sunitinib treatment (−30%, −70% and −66% in rats no.1, no. 2 and no. 3 respectively). Sunitinib treatment was maintained upto day 109 for rat no. 2 and up to day 116 for rats no. 1 and no. 3.Treatment with sunitinib for longer periods (up to 3 days for rat no. 2and 10 days for rats no. 1 and no. 3) resulted in a decrease in allmeasured parameters, tumor size, Imax and LPO.

Interestingly in rat no. 2, immunohistochemistry performed 3 days afterinitiation of sunitinib treatment, showed large area of necrosis in thetumor, in agreement with the reduction of all echographic parameters (asshown in FIG. 103).

Finally, in rat no. 4, which did not receive sunitinib,immunohistochemistry was performed on day 78 after NMU treatment. Tumorsize was around 1.5 cm². Staining for CD31, a marker of endothelialcells, revealed a strong staining of vessels (mostly straight) in stromatissue surrounding tumor nodules, whereas the staining was weak insidethe tumor nodules. Conversely KDR staining in tumor vessels was verystrong and showed tortuous vessels in tumor nodules, whereas only a fewvessels were stained for KDR in the stroma (as shown in FIG. 104).

Conclusion

These data indicate that use of targeted microbubbles comprising KDRbinding peptides such as D5, allows for the early evaluation of theefficacy of anti-angiogenic treatment therapies. Thus, according tocertain embodiments, the present invention allows for adjustments totherapeutic regimens to be made as needed and in a timely manner.

Materials and Analytical Methods for Examples 48-64

Solvents for reactions, chromatographic purification and HPLC analyseswere E. Merck Omni grade solvents from VWR Corporation (West Chester,Pa.). N-Methylpyrrolidinone (NMP) and N,N-dimethylformamide (DMF) wereobtained from Pharmco Products Inc. (Brookfield, Conn.), and werepeptide synthesis grade or low water/amine-free Biotech grade quality.Piperidine (sequencing grade, redistilled 99+%) and trifluoroacetic acid(TFA) (spectrophotometric grade or sequencing grade) were obtained fromSigma-Aldrich Corporation (Milwaukee, Wis.) or from the Fluka ChemicalDivision of Sigma-Alrich Corporation. N,N′-Diisopropylcarbodiimide(DIC), phenol (99%), N,N-diisopropylethylamine (DIEA) andtriisopropylsilane (TIS) were purchased from Sigma-Aldrich Corporation.Fmoc-protected amino acids, pseudoproline dipeptides,Fmoc-Asp(O-tBu)-Ser(ψMe,Mepro)-OH and Fmoc-Gly-Thr(ψMe,Mepro)-OH andN-hydroxybenzotriazole (HOBt) were obtained from Novabiochem (San Diego,Calif.). Fmoc-8-amino-3,6-dioxaoctanoic acid (Adoa) was obtained fromNeoMPS Corp (San Diego, Calif.) or Suven Life Sciences (Hyderabad,India). Disuccinimidyl glutarate (DSG) and1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethyleneglycol)2000]ammoniumsalt, [DSPE-PEG2000-NH2] were obtained from Pierce Chemical Co.(Rockford, Ill.) and Avanti® Polar Lipids (Alabaster, Ala.),respectively. Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH were preparedin-house from the corresponding triglycine or diglycine by the reactionwith Fmoc-OSu. An AG MP-50 ion-exchange resin was obtained from Bio-Rad(Hercules, Calif.).

Analytical HPLC data were generally obtained using a Shimadzu LC-10AT VPdual pump gradient system employing a Waters XTerra MS-C18 4.6×50 mmcolumn, (particle size: 5 μL; 120 Å pore size) and gradient or isocraticelution systems using water (0.1% TFA) as eluent A and CH3CN (0.1% TFA)or CH3CN—CH3OH (1:1, v/v) (0.1% TFA) as eluent B. Detection of compoundswas accomplished using UV at 220 and 254 nm. The purity of thephospholipid-PEG-peptide derivatives was determined on a YMC C-4 (5 μM,300 Å, 4.6×250 mm) column or on a Zorbax 300 SB-C3 (3.5 μM; 300 Å, 3×150mm) column using a SEDEX 55 Light Scattering Detector (LSD) and with aUV detector.

Preparative HPLC was conducted on a Shimadzu LC-8A dual pump gradientsystem equipped with a SPD-10AV UV detector fitted with a preparativeflow cell. Generally the solution containing the crude peptide wasloaded onto a reversed phase C18, C4 or C3 column, depending on thecompound characteristics, using a third pump attached to the preparativeShimadzu LC-8A dual pump gradient system. After the solution of thecrude product mixture was applied to the preparative HPLC column thereaction solvents and solvents employed as diluents, such as DMF orDMSO, were eluted from the column at low organic phase composition. Thenthe desired product was eluted using a gradient elution of eluent B intoeluent A. Product-containing fractions were combined based on theirpurity as determined by analytical HPLC and mass spectral analysis. Thecombined fractions were freeze-dried to provide the desired product.

Amino acid composition analyses were performed at the Keck BiotechnologyResource Laboratory at Yale University, New Haven, Conn. Mass spectraldata were obtained from MScan Inc. (606 Brandywine Parkway, West ChesterPa. 19380) or obtained in-house on an Agilent LC-MSD 1100 MassSpectrometer. For the purposes of fraction selection andcharacterization of the products mass spectral values were usuallyobtained using API-ES in negative ion mode. Generally the molecularweight of the target peptides was ˜3000; the mass spectra usuallyexhibited doubly or triply negatively charged ion mass values ratherthan [M-H]—. These were generally employed for selection of fractionsfor collection and combination to obtain the pure peptide during HPLCpurification. In some cases fractions exhibited dominant peaksattributable to [M-2H]/2+57 or [M-2H]/2+114 in the mass spectrum. Thesepeaks are due to the formation of adducts of one or two molecules oftrifluoroacetic acid per molecule of the peptide. After carefulcollection of fractions by comparing MS results and HPLC purities andfreeze-drying process, a small amount of the isolated fluffy solid wasdissolved in water (0.5 mg/mL) and treated with a drop of aqueousN-methyl-D-glucamine (˜0.5 M). This solution was analyzed by HPLC and MSfor final purity results of the purified peptide. Peptide solutions inthe presence of N-methyl-D-glucamine did not exhibit [M-2H]/2+57 or[M-2H]/2+114 mass value peaks in the mass spectrum, instead the expected[M-2H]/2 or [M-3H]/3 peaks were observed.

Examples 48-49 below refer to the monomeric peptide phospholipidconjugate shown in FIG. 106. A process for synthesizing this compound isshown in FIG. 105. Although these Examples refer more specifically tothe process for synthesizing the compound shown in FIG. 106, similarprocesses may used to prepare the monomeric peptide phospholipidconjugate shown in FIG. 114 and the linear peptide monomer (32) shown inFIG. 113 as well as other monomer peptide-phospholipid conjugatesincluding, for example, those formed using peptide monomersAc-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 623) andAc-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 624), as well asanalogs and derivatives of any of the above. Additionally, co-pendingU.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, sets forthmethods for the preparation of the peptide monomers and is incorporatedby reference herein in its entirety.

Example 48

Solid Phase Synthesis (SPPS) and Purification of Linear Peptide Monomer(2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2, (SEQ ID NO. 619)Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2,;N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide

The linear peptide monomer (2) was synthesized by an establishedautomated protocol on a SONATA®/Pilot Peptide Synthesizer usingFmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids andDIC-mediated HOBt ester activation in DMF. The peptide sequence wassynthesized in stepwise fashion by SPPS methods on the Fmoc-Pal-Peg-PSresin, typically on a 10 mmol scale. The amino acid couplings werecarried out with a 4-fold excess each of amino acid and the DIC-HOBtreagent pair in DMF.

In a typical coupling of an amino acid, 5 mL of dry DMF per gram ofresin was used. The total volume of DMF, calculated on the basis ofresin used, was allocated among amino acid, HOBt and DIC for solutionpreparation. For example, for the synthesis involving 50 g (10 mmolscale) of resin, the calculated volume of 250 mL of DMF was distributedamong amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acidvessel on the Sonata Pilot Peptide Synthesizer was charged with thesolid dry amino acid (4-fold excess with respect to the resin). Atinception of the coupling step, the software of the instrument wasemployed to deliver successively the chosen volume of DMF (for dilutionof the amino acid) and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF andmixing by nitrogen bubbling was initiated and conducted for 4 min. Thisserved to pre-activate the amino acid and to insure complete dissolutionof all components of the mixture. After activation, the softwaremediated the transfer of the solution of the activated Fmoc-amino acidto the reaction vessel containing the resin. After transfer was completethe vessel was agitated for 3 h with recurrent nitrogen bubbling. Afterthe 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g,6×) and the cleavage of the Fmoc-group was performed with 25% piperidinein DMF (5 mL/g) containing HOBt (0.1M) (2×10 min). The resin wasthoroughly washed with DMF (5 mL/g, 6×) to assure complete removal ofpiperidine from the resin in preparation for the ensuing amino acidcoupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, thepre-activation in the amino acid bottle was not conducted in order tominimize the formation of diketopiperazine during the activation time asdiscussed in the text. Therefore, in these two cases, the solutions ofamino acid, HOBt and DIC were added to the reaction vessel sequentiallyand the coupling process was conducted with ‘in situ’ activation.

After chain elongation was completed, the Fmoc group of the N-terminalamino acid was removed in the standard manner followed by the standardwash with DMF (vide supra). The N-terminal amino acid was then capped bytreatment with freshly prepared acetylation mixture (0.5M aceticanhydride, 0.125M DIEA and 0.015M HOBt in DMF/6 mL/g of resin), 2×20min. After completion of the peptide synthesis, the resin was treatedwith the cleavage cocktail, ‘Reagent B’(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g ofresin) for 4 h. The volatiles were removed and the paste thus obtainedwas triturated with ether to provide a solid which was washed with ether(3×) with intervening centrifugation (to compact the suspended solids inorder to allow decantation of the supernatant) and then dried undervacuum to provide the required peptide as an off-white solid. A 10 mmolscale synthesis of the linear peptide monomer (2) gave 33.82 g (103% oftheory) of the crude peptide. The greater than theoretical yield wasmost likely due to moisture and residual solvents.

Purification of the Linear Peptide Monomer (2)Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 619);Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2;N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide.

A ˜0.5 g portion of the crude linear peptide monomer (2) was dissolvedin a minimum amount of CH3CN (˜20 mL). The volume of the solution wasadjusted to ˜100 mL with water and employing a third pump the solutionwas loaded onto a reversed phase C18 preparative column (Waters, XTerra®Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) which had beenpre-equilibrated with 10% CH3CN in water (0.1% TFA). The column was noteluted with the equilibrating eluent during application of the samplesolution. After the sample solution was applied to the column, thecomposition of the eluent was ramped to 20% CH₃CN-water (0.1% TFA) over1 min, and a linear gradient at a rate of 0.6%/min of CH3CN (0.1% TFA)into water (0.1% TFA) was initiated and maintained for 50 min. Fractions(15 mL) were manually collected using UV at 220 nm as an indicator ofproduct elution. The collected fractions were analyzed on a WatersXTerra analytical reversed phase C-18 column (5 particle, 120 Å pore)and product-containing fractions of >95% purity were pooled andfreeze-dried to afford the corresponding pure linear peptide monomer(2). Typically the purification of 0.5 g of crude (2) afforded 0.12 g(24% yield) of the desired product (>95% purity).

Example 49

Preparation of Monomeric Peptide Phospholipid Conjugate (1)Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO.618);Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-(DSPE-PEG2000-NH-Glut)-NH2;N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-1-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide.

The monomeric peptide phospholipid conjugate (1),Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO.618), was prepared by conjugation of (3), the glutaric acid monoamidemono-NHS ester of peptide monomer (2), with DSPE-PEG2000-NH2phospholipid ammonium salt (4).

A round-bottomed flask equipped with magnetic stir bar and septum capwas charged sequentially with anhydrous dimethylformamide (7.5 mL),disuccinimidyl glutarate (DSG, 0.25 g, 0.75 mmol) anddiisopropylethylamine (0.10 g, 0.78 mmol) with stirring. Solid linearpeptide monomer (2) (0.5 g, 0.152 mmol) was added portionwise to theabove solution over a period of 2 min; then the solution was stirred for30 min at ambient temperature. The reaction mixture was diluted to ˜50mL with anhydrous ethyl acetate; this resulted in precipitation of theintermediate mono-NHS ester (3), the glutaric acid monoamide mono-NHSester of peptide monomer (2). The solution was centrifuged to bring downmono-NHS ester (3) as a colorless solid. The supernatant containingexcess DSG was decanted from the compacted solid mono-NHS ester (3)which was again dispersed in ethyl acetate, centrifuged and washed twicemore to remove the remaining traces of DSG. The solid intermediatemono-NHS ester (3) thus obtained was dissolved in anhydrous DMF (10.0mL); diisopropylethylamine (0.10 g, 0.78 mmol) was added; and themixture was stirred.

Meanwhile, DSPE-PEG2000-NH₂ phospholipid ammonium salt (4) (0.38 g, 0.14mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separateflask and trifluoroacetic acid (2 drops) was added to protonate thephosphodiester oxygen facilitating solubilization of phospholipidammonium salt in dichloromethane. The clear solution was then evaporatedon a rotary evaporator to remove the volatiles and dried further undervacuum.

The solid phospholipid ammonium salt (4) was dissolved in DMF (5 mL) andtransferred to the stirred solution of mono-NHS ester (3) and theresulting mixture was stirred for 24 h at ambient temperature. Thereaction mixture was diluted to 100 mL with a 1:1 mixture of CH₃OH andCH₃CN-water (1:1, v/v) and the insolubles were filtered. Half of thefiltered solution was loaded onto a reversed phase C2 preparative column(Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm) which had beenpre-equilibrated with 3:1 (v/v) mixture of water (0.1% TFA) andCH₃OH—CH₃CN (1:1, v/v, 0.1% TFA) at a flow rate of 100 mL/min. Note thatthe column was not eluted with the equilibrating eluent during loadingof the sample. After the sample solution was loaded the column waswashed with the equilibration eluent until the plug of DMF was eluted.The composition of the eluent was ramped to 70% CH₃OH—CH₃CN (1:1, 0.1%TFA) over 9 min and a linear gradient of 0.75%/min of CH₃OH—CH₃CN (1:1,0.1% TFA) into water (0.1% TFA) was initiated and run for 40 min.Fractions (15 mL) were collected using UV (220 nm) as an indicator ofproduct elution. Fractions were checked for purity on an analytical HPLCsystem (column: YMC C-4, 5μ, 300 Å, 4.6×250 mm) using UV at 220 nm andan evaporative light scattering detector (ELSD). The latter detector(ELSD) was employed to detect DSPE-PEG2000-NH2 phospholipid ammoniumsalt (4) which has very little UV absorbance at 220 nm.Product-containing fractions of >98% purity, and devoid ofDSPE-PEG2000-NH2 phospholipid ammonium salt (4) were combined andconcentrated on a rotary evaporator to reduce the content of CH₃OH. Theconcentrated solution was then diluted with 10% CH3CN in water until afaint flocculent precipitate formed. The resulting solution wasfreeze-dried to provide monomeric peptide phospholipid conjugate (1) asa colorless solid. The second portion of crude monomeric peptidephospholipid conjugate (1) was purified as described above. The combinedyield of the target monomeric peptide phospholipid conjugate (1) was0.40 g (47% yield).

Examples 50-52 below refer to the dimeric peptide phospholipid conjugateshown in FIG. 109. Representative methods of synthesizing the dimericconjugate are shown in FIGS. 107, 108, 110, 111 and 112.

Example 50

Solid Phase Synthesis (SPPS), Cyclization and Purification of MonomerPeptides (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH2 and (13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2

The linear peptides were synthesized by an established automatedprotocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PSresin (0.2 mmol/g), Fmoc-protected amino acids and DCI-mediated HOBtester activation in DMF. The peptide sequence on the Fmoc-Pal-Peg-PSresin was synthesized in stepwise fashion by SPPS methods typically on a10 mmol scale. The amino acid coupling was carried out with a 4-foldexcess each of amino acid and DIC-HOBt reagent in DMF.

In a typical coupling of an amino acid in the sequence, 5 mL of dry DMFper gram of resin was used. The total volume of DMF, calculated on thebasis of resin used, was allocated among amino acid, HOBt and DIC forsolution preparation. For example, for the synthesis involving 50 g ofresin, the calculated volume of 250 mL of DMF was distributed amongamino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vesselon the Sonata® Pilot Peptide Synthesizer was charged with the solid dryamino acid (4-fold excess with respect to the resin). At inception ofthe coupling step, the chosen volume of DMF and HOBt (4 eq.) in DMF andDIC (4 eq.) in DMF were delivered successively and after each deliverymixing by nitrogen bubbling was conducted. After the last reagent wasdelivered mixing by nitrogen bubbling was initiated and conducted for 4min. This served to preactivate the amino acid and to insure completedissolution of all components of the mixture.

After activation, the solution of the activated Fmoc-amino acid wastransferred to the reaction vessel containing the resin. After transferwas complete the vessel was agitated for 3 h with recurrent nitrogenbubbling. After the 3 h coupling time, the resin was washed thoroughlywith DMF (5 mL/g, 6×) and the cleavage of the Fmoc-group was performedwith 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2×10 min).The resin was thoroughly washed with DMF (5 mL/g, 6×) to assure completeremoval of piperidine from the resin in preparation for the ensuingamino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH andFmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was notconducted in order to minimize the formation of diketopiperazine duringthe activation time as discussed in the text. Therefore, in these twocases, the solution of the amino acid, HOBt and DIC were added to thereaction vessel sequentially and the coupling process was conducted with‘in situ’ activation. After chain elongation was completed, the fmocgroup of the N-terminal amino acid was removed in the standard mannerfollowed by the standard wash with DMF (vide supra). The N-terminalamino acid was then capped by treatment with freshly preparedacetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBtin DMF-6 mL/g of resin), 2×20 min.

Functionalization of the ε-amino group of C-terminal Lysine moieties ofthe monomer peptides (with Fmoc-Adoa or with Fmoc-Lys(ivDde) asrequired) was accomplished by first removing the ivDde group of theε-amino group with freshly prepared 10% hydrazine in DMF (5 mL/g ofresin—2×10 min). For appending of Fmoc-Adoa or Fmoc-Lys(ivDde) thecoupling time was increased to 10 h. After completion of the peptidesynthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g ofresin) for 4 h. After evaporation of the volatiles under vacuum, thepaste was triturated with ether to provide a solid which was collectedby filtration washed with diethyl ether and dried. A 10 mmol scalesynthesis of (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]—NH₂ gave 30 g(103% of theory) of the crude peptide. In the case of (13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂, a 10 mmol scale synthesisgave 28 g (107% of theory) of crude peptide. The greater thantheoretical yields are most likely due to moisture and residualsolvents.

Cyclization of the Linear Di-Cysteine Peptides to Cyclic DisulfidePeptides

Cyclic disulfide peptides were prepared from the corresponding lineardi-cysteine peptides by DMSO-assisted oxidation using DMSO/water (95/5,v/v). The crude linear peptide was dissolved in the solvent mixture (5mL/g) in a wide mouth beaker, and the pH of the solution was adjusted to8.5 by the addition of solid N-methyl-D-glucamine in portions. Theresulting mixture was stirred for 36 h at ambient temperature. Thesolution was then diluted with acetonitrile (50 mL/g) and the mixturewas stirred for 2 min. The solid cyclic disulfide peptide was collectedby filtration, washed with diethyl ether and dried.

Purification of Monomer Peptides

Peptide Monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH₂;Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Lys(ivDde)]-NH₂cyclic (6-13) disulfide

A ˜0.5 g portion of the crude cyclic disulfide peptide monomer (12) wasdissolved in a minimum amount of DMSO (˜3 mL). The volume of thesolution was adjusted to ˜100 mL with 20% CH₃CN-water and employing athird pump, the solution was loaded onto a reversed phase C18preparative column (Waters, XTerra® Prep MS C18, 10μ, 300A, 50×250 mm,flow rate 100 mL/min), which had been pre-equilibrated with 10% CH₃CN inwater (0.1% TFA). During application of the sample solution to thecolumn the flow of the equilibrating eluent from the preparative HPLCsystem was stopped. After the sample solution was applied to the column,the flow of equilibrating eluent from the gradient HPLC system wasreinitiated and the column was eluted with 10% CH₃CN-water (0.1% TFA)until the DMSO was eluted. Then the eluent composition was ramped to 35%CH₃CN-water (0.1% TFA) over 1 min after which a linear gradient at arate of 0.5%/min CH₃CN (0.1% TFA) into water (0.1% TFA) was initiatedand maintained for 50 min. Fractions (15 mL) were manually collectedusing UV at 220 nm as an indicator of product elution. The collectedfractions were analyzed on a Waters XTerra analytical reversed phaseC-18 column (5μ particle, 120 Å pore) and product-containing fractionsof >95% purity were pooled and freeze-dried to afford the correspondingcyclic disulfide peptide monomer (12). Typically the purification of 0.5g of crude peptide monomer (12) afforded 0.1 g (20% yield) of thedesired product (>95% purity).

Peptide Monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂;Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH₂cyclic (2-12) disulfide

Following the procedure employed for the HPLC purification of peptidemonomer (2), the crude cyclic disulfide peptide monomer (13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 (0.5 g) dissolved in 20%CH₃CN-water mixture (100 mL) was loaded onto a reversed phase C18preparative column (Waters, XTerra® Prep MS C18, 50×250 mm, 10μparticle, 300 Å pore, flow rate 100 mL/min), which had beenpre-equilibrated with 10% CH₃CN (0.1% TFA) in water (0.1% TFA). Duringapplication of the sample solution to the column the flow of theequilibrating eluent from the preparative HPLC system was stopped. Afterthe sample solution was applied to the column, the flow of equilibratingeluent from the gradient HPLC system was reinitiated and the column waseluted with 10% CH₃CN-water (0.1% TFA) for 5 min. Then the eluentcomposition was ramped to 30% CH₃CN (0.1% TFA)-water (0.1% TFA) over 1min and a linear gradient elution at a rate of 0.5%/min of CH₃CN (0.1%TFA) into water (0.1% TFA) was initiated and maintained for 50 min.Fractions (15 mL) were manually collected using UV at 220 nm as anindicator of product elution. The fractions were analyzed on a WatersXTerra analytical reversed phase C-18 column (4.6 mm i.d.×50 mm, 5μparticle, 120 Å pore) and product-containing fractions of >95% puritywere pooled and freeze-dried to afford the corresponding cyclicdisulfide peptide monomer (13). Typically the purification of 0.5 g ofcrude peptide monomer (3) afforded 0.12 g (24% yield) of the desiredproduct (>95% purity).

Example 51

Preparation and Purification of Precursor Dimer Peptide (16)Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide;Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-Adoa-Adoa-Glut-Lys)]-NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide

As shown in FIG. 107, disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol)was dissolved in stirred anhydrous dimethylformamide (2.0 mL) anddiisopropylethylamine (0.11 g, 0.85 mmol) was added in one portion. Thensolid peptide monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH₂(0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSGover a period of two min. After stirring for 30 min at room temperature,the solution was diluted with anhydrous ethyl acetate to ˜50 mL, (thisserved to precipitate intermediate mono-NHS ester (14)). The entiremixture was centrifuged and the supernatant was decanted leavingintermediate mono-NHS ester (14) as a colorless solid. The solid wasresuspended with ethyl acetate; the solution containing the suspendedsolid mono-NHS ester (14) was centrifuged to separate the solid and thesupernatant was again decanted. This washing process was repeated twiceto remove completely the excess DSG.

The solid mono-NHS ester (14) was dissolved in stirred anhydrousdimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol)was added. Then solid peptide monomer (13),Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂, (0.50 g, 0.19 mmol, 1.12 eq.)was added in portions to the stirred solution over a three min. periodand the resulting mixture was stirred for 18 h. The reaction wasmonitored by mass spectrometry; after the complete consumption of thepeptide monomer glutaric acid monoamide mono-NHS ester (14) wasconfirmed, neat hydrazine (0.1 mL) was added to remove the ivDdeprotecting group of the ivDde-bearing dimer (15) and the mixture wasstirred for 20 min at room temperature.

The solution was then acidified by dropwise addition of TFA and themixture was diluted to 100 mL with 10% CH₃CN (0.1% TFA) in water (0.1%TFA). The solution was filtered to remove particulates and half of theclarified solution was loaded onto a reversed phase C18 preparativecolumn (Waters, XTerra® Prep MS C18, 10μ, 50×250 mm, flow rate 100mL/min) pre-equilibrated with 10% CH₃CN in water (0.1% TFA). Duringapplication of the sample solution to the column the flow of theequilibrating eluent from the preparative HPLC system was stopped. Afterthe sample solution was applied to the column, the flow of equilibratingeluent from the gradient HPLC system was reinitiated and the column waseluted with 10% CH₃CN-water (0.1% TFA) in order to flush DMF from thecolumn. After elution of the DMF plug was completed the eluentcomposition was increased to 20% CH₃CN over one min. and the elution wascontinued with a linear gradient rate of 0.6%/min of CH₃CN (0.1% TFA)into water (0.1% TFA). Fractions (15 mL) were collected using UV (220nm) as an indicator of product elution. The fractions were analyzed on areversed phased C18 column (Waters MS C18, 4.6 mm i.d.×50 mm, 5μparticle, 120 Å pore) and the product-containing fractions of >95%purity were pooled and freeze-dried to provide precursor dimer peptide(16) as a colorless, fluffy solid. The remaining crude precursor dimerpeptide (16) was purified in the same manner. From 0.5 g each of monomerpeptides (12) and (13), 320 mg (overall yield 33%) of the desired dimer(16) was obtained (>95% purity).

Example 52

Preparation of KDR-Binding Dimeric Peptide Phospholipid Conjugate (11)Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amidecyclic (2-12) disulfide]-amide cyclic (6-13) disulfide;Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH₂cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide;Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys{Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys[-Adoa-Adoa-Glut-Lys(DSPE-PEG2000-NH-Glut)-]-NH₂cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide

The KDR-binding dimer (11) may be prepared by conjugation of precursordimer peptide (16),Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide, withDSPE-PEG2000-NH₂ phospholipid ammonium salt (18) as shown in FIG. 108.

Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was addedportionwise to a solution of disuccinimidyl glutarate (DSG, 0.15 g, 0.46mmol), and diisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF(3.0 mL) with stirring over a period of 3 min. Then the solution wasstirred at ambient temperature for 30 min. The reaction mixture wasdiluted to ˜50 mL with anhydrous ethyl acetate; this resulted inprecipitation of the dimer glutaric acid monoamide mono-NHS ester (17),the glutaric acid monoamide mono-NHS ester of the precursor dimerpeptide (16). The solution was centrifuged to pellet 6 (m/z, neg. ion,1887.3 (M-3H)/3, 1415.1 (M-4H)/4, 1131.9 (M-5H)/5) as a colorless solid.The supernatant ethyl acetate layer containing excess DSG was decantedfrom the compacted solid dimer glutaric acid monoamide mono-NHS ester(17) which was again resuspended in ethyl acetate, centrifuged andwashed twice more to remove the remaining traces of DSG. The solidintermediate glutaric acid monoamide mono-NHS ester dimer derivative(17) thus obtained was dissolved in anhydrous DMF/CH₂Cl₂ (8:2, v/v) (3.0mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and thesolution was stirred.

Meanwhile, DSPE-PEG2000-NH₂ phospholipid ammonium salt (18) (0.235 g,0.084 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in aseparate flask and TFA (2 drops) was added to protonate thephosphodiester oxygen, facilitating solubilization of phospholipidammonium salt (18) in dichloromethane. The clear solution wasconcentrated to remove the volatiles and dried further under vacuum.

The solid phospholipid ammonium salt (18) was dissolved in DMF (2 mL)and transferred to the stirred solution of glutaric acid monoamidemono-NHS ester dimer derivative (17) and the resulting mixture wasstirred for 24 h at ambient temperature. The reaction mixture wasdiluted with a solution of 50% CH₃OH, 25% CH₃CN and 25% water (1:1) to˜100 ml, and the insolubles were filtered. Half of the filtered solutionwas loaded onto a reverse phased C4 preparative column (Kromasil® PrepC4, 10μ, 300 Å, 50×250 mm) which had been pre-equilibrated with 1:1mixture of CH₃OH and CH₃CN (1:1, 0.1% TFA) and water (0.1% TFA) at aflow rate of 100 mL/min. During application of the sample solution tothe column the flow of the equilibrating eluent from the preparativeHPLC system was stopped. After the sample solution was loaded the flowof the equilibrating eluent was reinitiated and the column was washeduntil the plug of DMF was eluted.

The composition of the eluent was then ramped to 70% CH₃OH—CH₃CN (1:1,0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient of 0.75%/minof CH₃OH—CH₃CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated. Theelution was continued after reaching 100% B in order to achieve completeelution of the product from the column. Fractions (15 mL) were collectedusing UV (220 nm) as an indicator of product elution and after the mainproduct was eluted fraction collection was continued for several minutesin order to insure elution of trace amounts of starting phospholipidammonium salt (18). Fractions were checked for purity on an analyticalHPLC system (column: YMC C4, 5 μM, 300 Å, 4.6×250 mm) using UV at 220 nmand an evaporative light scattering detector (ELSD). The latter detectoris employed to detect DSPE-PEG2000-NH₂ which has a weak chromophore at220 nm. Product-containing fractions of >98% purity, and devoid ofDSPE-PEG2000-NH2 phospholipid ammonium salt (8) were combined andconcentrated to reduce the content of CH₃OH. The solution was thendiluted with 10% CH₃CN in water until a faint flocculent precipitateformed. The resulting solution was freeze-dried to afford the dimericpeptide phospholipid conjugate (11) as a colorless solid. The secondportion of crude dimeric peptide phospholipid conjugate (11) waspurified as described above. The combined yield of the target dimericpeptide phospholipid conjugate (11) was 0.39 g (57% yield). The samplesof the dimeric peptide phospholipid conjugate (11) made from differentsample purification runs were pooled together, dissolved intert-butanol-acetonitrile-water mixture and re-lyophilized to providethe dimeric peptide phospholipid conjugate (11) as a colorless, fluffysolid which was further dried under vacuum.

Examples 53-55 below refer to the preparation of the dimerpeptide-phospholipid conjugate shown in FIG. 109, wherein the dimericconjugate contains very low levels of TFA. FIGS. 110-112 illustrate themethods described in the Examples below.

Example 53

Preparation of Dimeric Conjugate Having Low TFA Levels Via the Use of aGlutaryl Linker

-   -   Preparation of (23), (26) and dimer peptide (27) acetate salt by        conversion of (22), (25) and dimer peptide 27 ● nTFA salts to        acetates by AG MP-50 ion-exchange resin

For compound (23) an AG MP-50 ion-exchange resin (1.5 meq/mL resin bed)was suspended in 20% of CH₃CN/H₂O. The suspension was packed in a 3×30cm glass column and the final volume was 150 mL. The column wasconnected to a pump and a conductivity meter. It was washed with 20% ofCH₃CN/H₂O at 17 mL/min flow rate until the conductivity was below 1μs/cm. Compound (22) (210 mg) was dissolved in 20% of CH₃CN/H₂O (80 mL)and the resulting solution was loaded to the column. The column waswashed again with the same eluent until its conductivity was below 1μs/cm. A gradient of NH₄OAc in 20% of CH₃CN/H₂O was applied at 200 mM,400 mM, 600 mM and 800 mM, 250 mL each. The compound came out at 600 mMNH₄OAc. The fractions were analyzed by HPLC and the ones containing thecompound were combined and lyophilized several times until the weight ofthe material was constant. 176 mg of the pure material (23) was obtainedas a white fluffy solid. The yield was 83.8%.

Additional parameters and results were as follows: HPLC: Ret. Time: 5.6min; Assay >98% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA);Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; 1441.7 [M-2H]/2, 960.9 [M-3H]/3. CE analysis(counter-ion % wt./wt.): TFA estimated to be 0.3%; acetate 1.1%.

For compound (26), following the same procedure for compound (23), 300mg of the peptide TFA salt (25) in 80 mL of water was loaded at 17mL/min. to a 150 mL of AG MP-50 column, which was washed with H₂O toconductivity of 1 μs/cm. The column was then washed with H₂O again afterloading, and the same step gradient of aqueous NH₄OAc into H₂O asemployed for the ion exchange of compound (23) was applied.Lyophilization of the combined fractions to a constant weight afforded200 mg of the acetate (26) as a white fluffy solid. The yield was 66.7%.

Additional parameters and results were as follows: HPLC: Ret. Time: 5.6min; Assay 97.0% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (0.1% TFA);Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; 1336.9 [M-2H]/2, 890.8 [M-3H]/3; CE analysis(counter-ion % wt./wt.): TFA estimated to be 0.4%; acetate 4.2%; ICanalysis (F %): 0.26.

For the dimer peptide (27) acetate salt, similar to the procedure forcompound (23), an AG MP-50 column (100 mL wet volume) was washed with30% CH₃CN/H₂O until the conductivity was below 1 μs/cm. Compound (27) asthe TFA salt, (120 mg in 80 mL of 30% of CH₃CN/H₂O) was loaded onto thecolumn and the column was washed with the same eluent until theconductivity was stable at 1 μs/cm. A step gradient of NH₄OAc 30% ofCH₃CN/H₂O into 30% of CH₃CN/H₂O was run as for compound (23) and thecompound was eluted at ca 600 mM NH₄OAc. The combined fractions werelyophilized and then relyophilized several times until the materialdisplayed a constant weight to provide 104 mg of the pure material (27)as an acetate salt. The yield was 86.7%.

Additional parameters and results were as follows: HPLC: Ret. time: 5.2min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (0.1% TFA);Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; 1816.3 [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5;CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.2%; acetate0.15%.

Preparation and Purification of the Dimer Peptide (27) Acetate Salt fromCompound (23) and Compound (26)

To a solution of disuccinimidyl glutarate (18 mg, 0.055 mmol) inanhydrous DMF (0.1 mL) was added a solution of compound (23) (61 mg,0.021 mmol) in 0.2 mL of anhydrous DMF dropwise (pH 8, neutralized byDIEA). The clear solution was stirred at RT for 0.5 h. HPLC and MSshowed the completion of the reaction. Solvent was removed in vacuo andEtOAc (8 mL) was added to precipitate the intermediate (24). The mixturewas centrifuged and decanted to remove excess glutarate. This EtOAcwashing was repeated 3 more times and the resulting solid was driedusing a stream of dry nitrogen. It was then dissolved in 0.3 mL ofanhydrous DMF. Compound (26), (56 mg, 0.021 mmol) was added and the pHof the solution was adjusted to 8 by addition of DIEA. The solution wasstirred for 16 h at room temperature after which by HPLC and MS analysisindicated completion of the reaction. A 30 μL aliquot of NH₂NH₂ wasadded and the mixture was stirred for 5 min to cleave the ivDde group.The reaction mixture was analyzed by HPLC and MS, which indicatedcomplete removal of the ivDde group.

Before purification of the dimer peptide (27) acetate, caution was takento carefully wash the whole preparative HPLC system including the columnwith TFA-free eluents, CH₃CN/H₂O/10 mM NH₄OAc. The crude reactionmixture was then applied to a reverse phase C-18 preparative column(Atlantis C-18, 5 μm particle, 100 Å pore, 30×150 mm, flow rate 30mL/min), pre-equilibrated with 15% B (A: 10 mM NH₄OAc in H₂O; B: 10 mMNH₄OAc in CH₃CN/H₂O, 9/1, v/v). The column was washed with the sameeluent until the DMF plug was eluted. The eluent composition wasincreased to 25% B over 2 min. and then ramped to 65% B over 40 min. Thefractions were analyzed on an analytical reverse phase C-18 column(Waters MS C-18, 4.6×50 mm, 5 μm particle, 100 Å pore, flow rate 3mL/min) and the product-containing fractions of >95% purity were pooledand freeze-dried to afford 25 mg of the dimer peptide (27) as itsacetate salt as a fluffy white solid. The yield was 21.8%.

Additional parameters and results were as follows: HPLC: Ret. time: 5.2min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (0.1% TFA);Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CEanalysis (counter-ion % wt./wt.): TFA estimated to be less than 0.2%;acetate 1.1%.

Example 54 FIG. 111

Preparation of Dimer Peptide-Phospholipid Conjugates Having Low TFALevels Via Ion Exchange Resin

Preparation and Purification of the Phospholipid Peptide Conjugate (21)as its Acetate Salt from Dimer Peptide (27) Acetate Salt

To a solution of disuccinimidyl glutarate-DSG (3.7 mg, 11.3 μmol) inanhydrous DMF (0.1 mL) was added a solution of neutralized dimer peptide(27) acetate salt, (15 mg, 2.75 mmol) in anhydrous DMF (0.2 mL),dropwise. The reaction solution was stirred at RT for 0.5 h. HPLCanalysis with a Waters Xterra C-18 column and MS showed the completionof the reaction. The solvent was evaporated and EtOAc (8 mL) was addedto precipitate the intermediate (28). The vessel containing theprecipitated intermediate (28) was centrifuged and the liquid layer wasdecanted. This procedure was repeated 3 times to remove the excess ofDSG. The solid was dried with a stream of dry nitrogen and thendissolved in 0.3 mL of anhydrous DMF. DSPE-PEG2000-NH₂ ammonium salt(29) (6.5 mg, 2.33 μmol) was added in solid form and the pH of themixture was adjusted to (28). The reaction mixture was stirred at RT for16 h. The mixture was analyzed by MS and HPLC with a Zorbax 300 SB-C3column and this indicated that the reaction was complete.

To minimize the potential contamination of the product with TFA, thecrude reaction mixture was purified by preparative HPLC equipped using anew Zorbax 300SB-C3 column (21.2×150 mm, 5μ particle) which had neverbeen exposed to TFA. The HPLC system was pre-washed by CH₃CN/H₂O/NH₄OAcextensively to remove traces of TFA. The reaction mixture was loadedonto the column which was pre-equilibrated with 20% B (A: 10 mM NH₄OAcin H₂O; B: 10 mM NH₄OAc in CH₃CN/H₂O, 9/1 v/v) at a flow rate of 30mL/min. The column was eluted at 30 mL/min with the same eluent untilthe plug of DMF was eluted. The eluent composition was then increased to40% B over 3 min and then ramped to 90% B over 50 min. The collectedfractions were analyzed on an analytical reverse phase C-3 column(Zorbax 300SB-C3, 3×150 mm, 3.5 μm particle, 300 Å pore, flow rate: 0.5mL/min), where detection was accomplished using UV at 220 nm and anevaporative light scattering detector (ELSD). The fractions containingthe pure product were pooled and lyophilized. A 6.5 mg portion of thefinal product (21) acetate salt was obtained. The yield was 33.0%.

Additional parameters and results were as follows: HPLC: Ret. Time: 13.3min; Assay >99% (area %); Column: Zorbax 300SB-C3, 3×150 mm, 3.5 μm, 300Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN/MeOH 1/1 (0.1% TFA);Elution: Initial condition: 60% B, linear gradient 60-90% B over 3 min;Flow rate: 0.5 mL/min; Detection: UV at 220 nm and ELSD; CE analysis(counter-ion % wt./wt.): % wt. TFA: 0.3%; % wt acetate 0.4%.

Example 55 FIG. 112

Preparation of Dimeric Conjugate Having Low TFA Levels Via SequentialPurification Using Zorbax C-3 RP Preparative HPLC and Sephadex G-25 GelPermeation Chromatography

Materials used and conditions for the analytical HPLC system include thefollowing: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle;Eluent A: H₂O(HPLC Grade with 0.1% TFA by volume); Eluent B: CH₃CN (0.1%TFA by volume). Elution: Initial condition: 50% B then a linear gradientof 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5 mL/min;Detection: UV at 220 nm. Ret. time: (Compound (21)): 6.77 min, Rt(lyso): 4.06 min.

Preparative Hplc Using Preparative Zorbax C-3 Column to Remove theLyso-Compound from (21)

The crude compound was loaded at a concentration of 30% eluent B.Materials used and conditions include: Conditions: Column: Waters Zorbax300SB C-3; 21.2 mm i.d.×150 mm; 3.5 μm particle; Eluents: Eluent A: H2O(HPLC Grade with 10 mM NH4OAc); Eluent B: CH3CN/H2O, 9/1 (final NH4OAcconcentration: 10 mM).

The composition of the eluent was then changed to 45% B over 2 min, thenthe column was eluted with a linear gradient of 45-100% B over 40 min;Flow rate: 30 mL/min; Detection: UV at 220 nm.

The crude compound (100 mg) was dissolved in 25 mL of a solution of 30%B. The preparative HPLC system was equilibrated at 30% B. The compoundwas loaded on to the Zorbax C-3 column. The mobile phase composition wasramped to 45% B over 2 min. A linear gradient from 45-100% B over 40 minwas used for the elution of (21). The product eluted between 26.5-33min.

The fractions that contained (21) were combined and lyophilized to givea white fluffy solid. This was dissolved in water-acetonitrile, thenlyophilized again. This provided 70 mg product devoid of thelyso-compound. The recovery was about 70%. After chromatography wascompleted, the system was washed with 95% B for 15 min at a flow rate of30 mL/min. The column was then washed with CH₃CN/H₂O (50/50, without TFAor buffer) for 30 min at a flow rate of 15 mL/min. The column was thenstored at room temperature for future use. Analytical HPLC confirmed theabsence of the lyso-compound in the isolated material. Further analysisconfirmed that no lyso-compound formed after 5 days at room temperature.The material still contained significant amounts (4.2 wt %) of TFA.

Removal of TFA from (21) by Gel Permeation Chromatography on SephadexG-25

A Sephadex G-25 column (100 g resin, bead size 20-80 μm, total gelvolume ˜500 mL, column height: 27 cm) was equilibrated with 4 L of 50 mMammonium bicarbonate. Then (21) (70 mg) was dissolved in 30 mL (finalvolume) of 60 mM ammonium bicarbonate in 10% aqueous acetonitrile. Thesolution was filtered and then loaded on to the Sephadex G-25 column.The column was eluted with 50 mM ammonium bicarbonate buffer withcollection of 10 mL fractions. The collected fractions were monitored byanalytical HPLC (UV detection at 220 nm). The results are provided inTable 4-A below.

TABLE 4-A Fraction Volume Compound present (by HPLC # (mL) analysis offraction) 1 10 No 3 10 No 6 10 No 9 10 No 12 10 No 15 10 No 18 10 No 1910 No 20 10 Yes 21 10 Yes 24 10 Yes 27 10 Yes 28 10 Yes 29 10 No

Fractions 20-28 were pooled and lyophilized. The lyophilized materialobtained was re-dissolved in a small volume of water and the solutionwas frozen and lyophilized to remove residual amounts of ammoniumbicarbonate. The final weight of the desired material was 58 mg. Therecovery was 83%.

To ascertain the effective removal of TFA, the sample was subjected toCE analysis for TFA and acetate ions. The TFA is clearly present in thestarting material (4.2%) according to the previous assay, while it ishardly detected (0.2%) after the gel permeation procedure. No acetateion was detected.

Analytical Data for (21) Obtained by Serial Zorbax C-3 Preparative HPLCand Sephadex G-25 Gel Permeation Chromatography

Materials used and conditions for collecting analytical data include:Fluorine analysis (IC by QTI): 751 ppm (0.15% TFA wt/wt); Mass Spectrum:Method: MALDI-TOF; Mode: Positive Ion; Average molecular weight detectedwas 8461 the typical PEG2000 mass distribution curve was observed. HPLC:System A: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle;Eluent A: Water (HPLC Grade with 0.1% TFA by volume); Eluent B:Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution:linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flowrate: 0.5 mL/min; Detection: UV at 220 nm. Ret time: 6.77 min; Area %:99.6%. System B: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μmparticle; Eluent A: Water (HPLC Grade with 0.1% TFA by volume); EluentB: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution:linear gradient of 50-90% B over 3 min then ramp to 100% B over 12 min.Flow rate: 0.5 mL/min; Detection: LSD; Ret: time: 13.98 min. Area %:99.3%.

Table 5-A below provides definitions for the abbreviations used and thesources of materials referred to in Examples 56-59.

TABLE 5-A DSPA.Na (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3-phosphosphatidic acid, sodium salt DPPG.Na (Genzyme) IUPAC:1,2-Dipalmitoyl-sn-glycero-3- phosphoglycerol, sodium salt DPPE(Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine DSPCDistearoyl-glycero-phosphatidylcholine (Genzyme) IUPAC:1,2-Distearoyl-sn-glycero-3-phosphocholine DSPG.Na (Genzyme) IUPAC:1,2-Distearoyl-sn-glycero-3- phosphoglycerol, sodium salt DSPE-PEG1000Distearoyl-glycero-phosphoethanolamine-N- methoxy(polyethyleneglycol)1000 (Avanti Polar) DSPE-PEG2000Distearoyl-glycero-phosphoethanolamine-N- methoxy(polyethyleneglycol)2000 (Avanti Polar) Stearate* Sodium Stearate (Fluka) PEG4000(polyethylene glycol) MW 4000 (Fluka) Mannitol (Fluka) *the acid form,i.e., stearic acid, can also be used in any of the microbubblepreparations herein.

Example 56 Preparation of Targeted Microbubbles with DSPC/DPPG EnvelopeExample 56A

383 mg of a mixture of DSPC/DPPG/ and the dimeric peptide phospholipidconjugate (11) shown in FIG. 109 (molar ratio 49.75/49.75/0.5,corresponding to 187.1, 176.4 and 19.8 mg of the three components,respectively) and PEG-4000 (22.6 g) were solubilized in 120 g of t-butylalcohol at 60° C., in a water bath. The solution was filled in vialswith 0.8 mL of solution each. The samples were frozen at −45° C. andlyophilized. The air in the headspace was replaced with a mixture ofC4F10/Nitrogen (50/50) and vials capped and crimped. The lyophilizedsamples were reconstituted with 5 mL of H2O per vial.

Example 56B

Example 56A was repeated using a mixture of DSPC/DPPG/ and the monomericpeptide phospholipid conjugate (31) shown in FIG. 114 (molar ratio49.5/49.5/1, corresponding to 182.8, 172.3 and 28.2 mg of the threecomponents, respectively).

Example 57 Preparation of Targeted Microbubbles with DPPE/DPPG EnvelopeExample 57A

An aqueous suspension of DSPE-PEG1000 (0.43 mg—0.24 μmole) and themonomeric peptide phospholipid conjugate (31) shown in FIG. 114 (3.0mg—0.5 μmole) was prepared in 500 μL of distilled water at 60° C. toobtain a micellar suspension.

Separately, DPPE (15.8 mg—22.8 μmoles) and DPPG (4.2 mg—5.7 μmoles) weredispersed in a solution of mannitol 10% in distilled water (20 mL) at70° C. for 20 minutes. The dispersion was then cooled to roomtemperature. Perfluoroheptane (1.6 mL) was emulsified in the aqueousphase using a high speed homogenizer (Polytron PT3000, probe diameter of3 cm) for 1 minute at 10500 rpm to obtain an emulsion.

The micellar suspension was added to the emulsion and the resultingmixture was heated at 60° C. for 1 hour under stirring. After cooling toroom temperature (1 hour), the obtained emulsion was divided in 4 mLfractions in 50 mL round bottom flasks. The emulsion was frozen at −45°C. for 5 minutes and freeze-dried at 0.2 mBar for 24 hours (Freeze-DrierChrist Beta 1-8K).

Before redispersion, the lyophilisate was exposed to an atmospherecontaining C4F10/nitrogen (50/50 by volume). The lyophilized product wasthen dispersed in a volume of water twice the initial one by gentle handshaking

Example 57B

An aqueous suspension of DSPE-PEG1000 (0.5 mg—0.27 μmole) and dimericpeptidephospholipid conjugate (11) shown in FIG. 109 (5.3 mg—0.63 μmole)was prepared in 500 μL of distilled water at 60° C. to obtain a micellarsuspension.

Separately, DPPE (15.8 mg—22.8 μmoles) and DPPG (4.2 mg—5.7 μmoles) weredispersed in a solution of PEG4000 10% in distilled water (20 mL) at 70°C. for 20 minutes. The dispersion was then cooled to room temperature.Perfluoroheptane (1.6 mL) was emulsified in the aqueous phase using ahigh speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1minute at 10000 rpm to obtain an emulsion.

The micellar suspension was added to the emulsion and the resultingmixture was heated at 80° C. for 1 hour under stirring. After cooling toroom temperature (1 hour), the obtained emulsion was washed once bycentrifugation (200 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of phospholipid. The separated pellet (containing emulsifiedmicrodroplets of solvent) was recovered and re-suspended with theinitial volume of a 10% PEG4000 aqueous solution.

The obtained emulsion was sampled into DIN8R vials (1 mL/vial). Thenvials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mBar for 12 hours with a final dryingstep at 30° C. and 0.1 mBar for 7 hours.

Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 byvolume) and sealed. The lyophilized product was redispersed in a volumeof water twice the initial one by gentle hand shaking

Example 58 Preparation of Targeted Microbubbles with DSPC/DSPA EnvelopeExample 58A

An aqueous suspension of DSPE-PEG1000 (2.5 mg—1.4 μmole) and dimericpeptide conjugate (11) shown in FIG. 109 (7.0 mg—0.84 μmole) wasprepared in 1 mL of distilled water at 60° C. to obtain a micellarsuspension.

DSPC (16.3 mg—20.6 μmoles) and DSPA (3.7 mg—5.15 μmoles) were dissolvedin cyclooctane (1.6 mL) at 80° C. This organic phase was added to aPEG4000 10% solution in water (20 mL) using a high speed homogenizer(Polytron T3000, probe diameter of 3 cm) for 1 minute at 8000 rpm, toobtain an emulsion.

The micellar suspension was mixed with the emulsion and the resultingmixture was heated at 80° C. for 1 hour under agitation. After coolingto room temperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended intwice the initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DIN8R vials (1 mL/vial). Thenvials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours.

Vials were exposed to an atmosphere containing C4F10/Nitrogen (35/65 byvolume) and sealed.

The lyophilized product was then dispersed in a volume of water twicethe initial one by gentle hand shaking

Example 58B

Example 58A was repeated, but using 0.7 mg of DSPE-PEG2000 (0.26 μmoles)and 1.6 mg of monomeric peptide-phospholipid conjugate (1) shown in FIG.106 (0.26 μmole) to prepare the micellar suspension.

Example 58C

DSPC (16.3 mg—20.6 μmoles), DSPA (3.7 mg—5.15 μmoles) and monomericpeptide phospholipid conjugate (1) shown in FIG. 105 (1.6 mg—0.26 μmole)were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase wasemulsified in a PEG4000 10% aqueous phase (20 mL) using a high speedhomogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at8000 rpm to obtain an emulsion.

The resulting emulsion was heated at 80° C. for 1 hour under stirring.After cooling to room temperature (1 hour), the obtained emulsion wasdiluted with 20 ml of a PEG4000 10% aqueous solution.

The emulsion was sampled into DIN8R vials (1 mL/vial). Then vials werecooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-driedat −25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C.and 0.1 mBar for 7 hours.

Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 byvolume) and sealed. The lyophilized product was redispersed in a volumeof water twice the initial one by gentle hand shaking

Example 59 Preparation of Targeted Microbubbles with DSPC/StearateEnvelope Example 59A

An aqueous suspension of DSPE-PEG2000 (2.5 mg—0.9 μmoles) and thedimeric phospholipid conjugate (11) shown in FIG. 109 (2.5 mg—0.3μmoles) was prepared in 6604 of distilled water at 60° C. to obtain themicellar suspension.

Separately, DSPC (18.2 mg—23.1 μmoles) and stearate (1.8 mg—5.8 μmoles)were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase wasadded to a PEG4000 10% solution in water (20 mL) using a high speedhomogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at9000 rpm, to obtain an emulsion.

The micellar solution was mixed with the emulsion and the resultingmixture was heated at 80° C. for 1 hour under agitation. After coolingto room temperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of phospholipids. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended withtwice the initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DIN8R vials (1 mL/vial). Thenvials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours.

Vials were exposed to an atmosphere containing C4F10/Nitrogen (35/65 byvolume) and sealed.

The lyophilized product was dispersed in a volume of water twice theinitial one by gentle hand shaking

Example 59B

Example 59A was repeated by replacing the dimeric peptide phospholipidconjugate (11) shown in FIG. 109 with the same relative molar amount ofthe monomeric peptide phospholipid conjugate (1) shown in FIG. 106.

Example 59C

Example 58C was repeated with DSPC (18.2 mg—23.1 μmoles), sodiumstearate (1.8 mg—5.8 μmoles) and the dimeric peptide phospholipidconjugate (11) shown in FIG. 109 (2.2 mg—0.26 μmole). The agitationspeed for emulsification was fixed to 9000 rpm. After cooling to roomtemperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended intwice the initial volume of a 10% PEG 4000 aqueous solution.

Example 60 Static Binding Test on KDR-Transfected Cells

Plasmid Production and Purification

Full-length KDR was cloned into the pcDNA6 vector and the plasmid wasamplified in competent DH5α E. coli. Plasmid amplification andpurification was performed using E. coli JM 109 and a kit from Quiagen.

Transfection of 293H Cells on Thermanox® Coverslips

Cells were grown on poly-D-lysine-coated Thermanox® circular coverslipsin 24-well plate. Transfection was done as recommended in thelipofectamine 2000 protocol (Invitrogen, cat#11668-019) using 1 μg ofDNA (pc-DNA6-fKDR)/per coverslip (1.3 cm2) in 0.1 mL. Transfection wasdone in serum-free media, the transfection reagent mix was removed fromcells after 2 hours and replaced with regular serum-containing medium.Some of the cell-coated coverslips were mock-transfected (with no DNA).The next day, expression of the KDR receptor was assessed byimmunocytochemistry and the binding assay was performed.

Bubble Binding Assay

The transfected cells were incubated with KDR-targeted microbubblesresuspended in 50% human plasma in PBS. For the incubation with thetransfected cells a small plastic cap was filled with a suspensioncontaining a 1.3×108 bubbles and the cap was covered with an invertedThermanox® coverslip so as to put the transfected cells in contact withthe targeted microbubbles. After 30 min of incubation at RT, thecoverslip was lifted with tweezers, rinsed three times in PBS andexamined under a microscope to assess binding of the targetedmicrobubbles.

Determination of the % of Surface Covered by Microbubbles

Images were acquired with a digital camera DC300F (Leica) and thepercent of surface covered by bound microbubbles in the imaged area wasdetermined using the software QWin version 3.1 (Leica Microsystem AG,Basel, Switzerland). Pictures were taken of each Thermanox® coverslip.For each preparation of Examples 56 and 57, the binding assay wasrepeated a minimum of two times thus obtaining an average value of thesurface covered. In the following Tables 6-A and 7-A, the bindingactivity of the microbubbles prepared according to Examples 56 and 57above are recorded.

As indicated by the Tables, the same peptide may show different bindingactivities when included (as a lipopeptide) in different phospholipidformulations forming the stabilizing envelope of the microbubble.Microbubbles containing KDR binding lipopeptides of the invention bindspecifically to KDR-expressing cells while they did not bind appreciablyto mock transfected cells.

Example 61 Dynamic Binding test on rh VEGF-R2/Fc Chimeric Protein

Preparation of Fc-VEGF-R2-Coated Coverslips

Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, Pa., USA)were coated with recombinant human VEGF-R2/Fc Chimeric protein (R&DSystems) according the following methodology.

A surface of dimensions 14×25 mm was delimited on the glass coverslipusing a special marker (Dako Pen) and 400 μL of Fc-VEGF-R2 solution at 4μg/mL in PBS was deposited on this surface. After an overnightincubation at 4° C., the solution was aspirated, replaced by 0.5 mL of asolution of BSA 1% w/v in PBS-0.05% Tween 80, pH 7.4 and incubated for 3hours at RT. Then the coverslip was washed three times with 5 ml ofPBS-0.05% Tween 80.

Binding Assay

Binding studies of targeted bubbles were carried out using aparallel-plate flow chamber (FCS2, Bioptech Inc., Butler, Pa., USA) witha chamber gasket of 0.25 mm in thickness, with a customized adapter forupside-down chamber inversion. The coated coverslip was inserted as aplate of the flow chamber. Microbubbles (5×106 bubbles/mL in 50% humanplasma in PBS) were drawn through the flow chamber using an adjustableinfusion pump (Auto Syringe® AS50 Infusion Pump, Baxter, Deerfield,Ill., USA) with a 60 mL syringe (Terumo). The pump flow rate wasadjusted to 1 mL/min to obtain the desired shear rate of about 114 s-1.After 10 minutes, the flow was stopped and pictures were taken randomlyat different positions on the coverslip (on areas of about 0.025 mm2)using a 40× objective and a CCD monochrome camera (F-View II, SoftImaging Systems, Germany) connected to an inverted Olympus IX 50microscope.

The number of microbubbles on each picture was determined, averaged withrespect to the total number of pictures and the obtained value was thendivided by ten (to obtain the “slope”, i.e. the average amount of boundmicrobubbles per minute).

For each preparation of Examples 58 and 59, the binding assay wasrepeated four times thus obtaining an average value of the slope.

The slope represents the bubble binding rate on the target substrate.For instance, a slope value of 8 indicates that an average of eighty(80) microbubbles was bound on the coated coverslip in ten minutes. Ahigher slope indicates a better capacity of bubbles to bind to thetarget under flow conditions.

In the following tables 8-A and 9-A, the binding activity of themicrobubbles prepared according to Examples 58 and 59 above wereillustrated.

As inferable from the tables, the same peptide may show differentbinding activities when included (as a peptide-phospholipid conjugate orlipopeptide) in different phospholipid formulations forming thestabilizing envelope of the microbubble.

TABLE 6-A KDR- Example KDR Mock Mock 56A 28.6% 0.4% 28.3% 56B 28.0% 0.3%27.7%

TABLE 7-A KDR- Example KDR Mock Mock 57A 23.6% 0.2% 23.5% 58B 28.0% 0.0%28.0%

TABLE 8-A Example Slope 58A 8.2 58B 8.1 58C 5.8

TABLE 9-A Example Slope 59A 9.0 59B 8.0 59C 7.8

Example 62 In Vivo Evaluation of Ultrasound Contrast Agents Targeted toKDR

The ability of ultrasound contrast agents containing KDR bindinglipopeptides of the invention to bind to KDR-expressing tissue in vivowas assessed using a known model of angiogenesis: the rabbit VX2 tumormodel.

A known model of angiogenic tissue was used to examine the ability ofthe KDR-targeted ultrasound microbubbles to localize to and provide animage of angiogenic tissue. The VX2 rabbit carcinoma was seriallyimplanted in the dorsal muscle of New Zealand rabbits (Charles RiverLaboratories, France) weighting 2.5/3 kg.

Preparation of Tumor Homogenate

Tumor was surgically removed, placed into McCoy's culture mediumcontaining 10% fetal calf serum, antibiotics, 1.5 mM Glutamax I and cutinto small pieces that were rinsed to remove blood and debris. Thentumor pieces (3 to 5 cm3) were placed in a 50 ml Falcon tube containing5 mL of complete medium. The tumor tissue was ground (Polytron) until nomore solid pieces were visible. The murky fluid was centrifuged for 5minutes at 300 g and the supernatant discarded. Seven mL of fresh mediumwas added per 5 mL of pellet.

Tumor Implantation

Rabbits received first 0.3 mL of Vetranquil (Acepromazine, Sanofi,injected intramuscularly) and were then anesthetized with anintramuscular injection of Ketaminol®5/Xylazine (Veterinaria AG/Sigma)mixture (50/10 mg/mL, 0.7 mL/kg). One hundred microliters of VX2 tumorhomogenate was injected intramuscularly. Fifteen days after implantationof VX2 tumors, animals were anesthetized again with the same mixture,plus subcutaneous injection of 50% Urethane (2 mL/kg, s.c.) (Sigma) forimaging experiments.

In Vivo Ultrasound Imaging

VX2 tumor imaging was performed using an ultrasound imaging system ATLHDI 5000 apparatus equipped with a L7-4 linear probe. B-mode pulseinversion at high acoustic power (MI=0.9) was used to evaluateaccumulation of targeted microbubbles on the KDR receptor expressed onthe endothelium of neovessels. The linear probe was fixed on the skindirectly over the implanted tumors.

After bubble injection (0.14/kg of gas) using the preparations of eitherExample 63 or Example 64, insonation was stopped allowing bubbles toaccumulate for 25 minutes. Then, insonation was reactivated at highacoustic power (MI 0.9) destroying all the bubbles present in the tumor.The amount of free circulating bubbles was then assessed by recordingthe signal obtained after 20 sec accumulation without insonation.

Video frames from VX2 tumor imaging experiments were captured withvideo-capture and analysed with Image-Pro Plus 2.0 software. The imagerepresenting free circulating bubbles was subtracted from the imageobtained at 25 min, to provide an image representing bound bubbles.

Referring to FIG. 115 (which shows the results with the preparation ofExample 63) and FIG. 116 (which shows the results with the preparationof Example 64), FIGS. 115A and 116A show an image before bubbleinjection (baseline); FIGS. 115B and 116B show retention of bubblecontrast in the tumor 25 minutes post injection; and FIGS. 115C and 116Cshow the result obtained after subtraction of the baseline and freecirculating bubbles and represent bound microbubbles containing KDRlipopeptides according to the present invention. Examples 62-64 andFIGS. 115 and 116 confirm that ultrasound contrast agents bearing suchKDR binding moieties localize to KDR expressing (and thus angiogenic)tissue in animal models.

Example 63

Example 59A was repeated by replacing DSPE-PEG2000 with DSPE-PEG1000(2.7 mg, 1.54 μmol) and using 2.5 mg (0.31 μmol) of dimeric peptidephospholipid conjugate (11) shown in FIG. 109.

Example 64

Example 63 was repeated by replacing the dimeric peptide phospholipidconjugate with the same molar amount of monomeric phospholipid conjugate(1) shown in FIG. 106.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. The publications, patents and otherreferences cited herein are incorporated by reference herein in theirentirety.

What is claimed is:
 1. A method for evaluating the effectiveness of atreatment protocol comprising: (a) administering an ultrasound contrastagent comprising a gas filled microvesicle and one or more KDR bindingpeptides selected from the group consisting of: AGPKWCEEDWYYCMITGTGGGK,(SEQ ID NO: 264) GDSRVCWEDSWGGEVCFRYDPGGGK, (SEQ ID NO: 294)AQDWYYDEILSMADQLRHAFLSGGK, (SEQ ID NO: 310) AGPTWCEDDWYYCWLFGTGGGK,(SEQ ID NO: 277) AGDWWVECRVGTGLCYRYDTGTGGGK, (SEQ ID NO: 286)AGPTWCADDWYYCWLFGTGGGK, (SEQ ID NO: 390) and VCWEDSWGGEVCFRYDPGGGK,(SEQ ID NO 337)

or a KDR-binding dimer selected from the group consisting of D5, D6, andD23, to a subject with a cell hyperproliferation or angiogenesisdisorder associated with KDR expression; (b) obtaining an initial imageof a KDR expressing cell or tissue in the subject; (c) administering atherapeutic agent appropriate for the treatment of the disorder; (d)obtaining a subsequent image of said KDR expressing cell or tissue inthe subject; and (e) comparing the initial and the subsequent images todetermine the increase or decrease in KDR binding, thereby evaluatingthe effectiveness of the treatment therapy.
 2. The method of claim 1,wherein the disorder is cancer.
 3. The method of claim 1, wherein theultrasound contrast agent comprises a dimer.
 4. The method of claim 3,wherein the dimer is D5.
 5. The method of claim 1, wherein the initialand subsequent images are obtained via diagnostic ultrasound.
 6. Themethod of claim 1, wherein the therapeutic agent is an anti-canceragent.
 7. The method of claim 6, wherein the cancer is selected from thegroup consisting of prostate cancer, mammary cancer, ovarian cancer,liver cancer, colon cancer, renal cancer, bone cancer, bladder cancer,pancreatic cancer, lung cancer, uterine cancer and testicular cancer. 8.The method of claim 2, wherein comparing the initial and subsequentimages to evaluate the effectiveness of therapy comprises comparing thesize or vascularity of a tumor shown in the images of the subject. 9.The method of any of claim 2 or 4, wherein comparing the initial andsubsequent images to evaluate the effectiveness of therapy comprisescomparing a parameter selected from the group consisting of Imax(maximal peak enhancement values), AUC (area under the curve), WIR(Wash-in-rate), and TTP (Time-to Peak).
 10. The method of any of claim 2or 4, wherein comparing the initial and subsequent images to evaluatethe effectiveness of therapy comprises comparing LPO (late phaseopacification).